KR101781120B1 - Composition for preventing or treating trail-resistant cancer comprising narcissus tazetta extracts or fraction thereof, and trail protein - Google Patents

Abstract

The present invention relates to a daffodil extract or its fractions, a pharmaceutical composition for preventing or treating TRAIL-resistant cancer containing TRAIL protein as an active ingredient, or a health functional food composition for preventing or improving TRAIL-resistant cancer.
The extract of the present invention or its fractions exhibits an effect of increasing the sensitivity of TRAIL-resistant cancer cells to cytotoxicity of TRAIL protein and inducing apoptosis of cancer cells. Therefore, Prevention, improvement, or cure.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating TRAIL-resistant cancer, which contains TRAIL protein as an active ingredient, and a composition for preventing or treating TRAIL-resistant cancer,

The present invention relates to a daffodil extract or its fractions, a pharmaceutical composition for preventing or treating TRAIL-resistant cancer containing TRAIL protein as an active ingredient, or a health functional food composition for preventing or improving TRAIL-resistant cancer.

Cancer is one of the most deadly diseases worldwide, and the cancer age is gradually decreasing, while the average life expectancy is gradually increasing, and cancer incidence is expected to increase further. According to the National Cancer Center's 2013 statistics (National Cancer Center, Cancer Facts & Figures 2013), p 18, 2013.) The number of cancer patients in Korea is 202,053, which is estimated to reach about 270,000 by 2015.

On the other hand, tumor necrosis factor-related apoptosis-inducing ligand (Tumor necrosis factor (TNF) -ralated apoptosis-inducing ligand; TRAIL] is a transmembrane protein that is known to be involved in a sub-apoptotic pathway by binding to membrane-binding death receptors that deliver apoptosis signals through the intracellular killing domain. In some cell lines, cell susceptibility to TRAIL is dependent on the expression of the cell membrane TRAIL receptor and caspase-8. Caspase-8 is activated in response to TRAIL and released into the cytoplasm where the protease cascade is initiated, which activates the operative caspases, including caspase-3 and -7.

Conventionally, the development of a novel cancer treatment method using TRAIL or TRAIL gene has been attempted. For example, Korean Patent Application No. 10-2003-7015417 discloses an antibody that immunospecifically binds to a TRAIL receptor, Patent Application No. 10-2004-0006450 discloses a method for inhibiting angiogenesis using TRAIL.

However, there have been several reports that many tumor cells, including human gastric adenocarcinoma (AGS) cell lines, are resistant to the apoptotic effects of TRAIL. Modification to the proapoptotic protein Bax and increased expression of IAP group factors may contribute to resistance to TRAIL-mediated apoptosis. Recently, it has been shown that chemotherapeutic agents sensitize TRAIL-mediated synergistic cytotoxicity in TRAIL-resistant cells. In conclusion, it is becoming increasingly important to investigate the anticancer drugs that can maximize the therapeutic effect of TRAIL-resistant cancer cells by overcoming such resistance.

Under these circumstances, the inventors of the present invention conducted a study to develop an anticancer composition that effectively works against TRAIL-resistant cancers, and thus the natural product, a dernicillin extract or its fractions, is sensitive to TRAIL cytotoxicity in TRAIL- To increase the cell death by apoptosis, and to accomplish a composition for preventing, ameliorating or treating cancer using the same.

One object of the present invention is the use of narcissus There is a tazetta . The present invention also provides a method for preventing or treating cancer comprising administering to a subject a pharmaceutical composition for preventing or treating TRAIL-resistant cancer, which comprises a chinensis extract or a fraction thereof and TRAIL protein as an active ingredient will be.

It is another object of the present invention to provide a health functional food composition for preventing or ameliorating TRAIL-resistant cancer, which contains the extract of Daffodil or its fraction and TRAIL protein as an active ingredient.

Yet another object of the present invention is to provide a feed additive, feed, drinking water additive or drinking water for preventing or improving TRAIL-resistant cancer, which contains the extract of the narcissus or its fraction and TRAIL protein as an active ingredient.

One aspect of the present invention is to provide a pharmaceutical composition for preventing or treating Narcissus tazetta var. Chinensis extract or fractions thereof and a TRAIL-resistant cancer containing TRAIL protein as an active ingredient, to provide.

In the present invention "narcissus (Narcissus There is a tazetta . chinensis ") is a perennial plant belonging to the family Amaryllidaceae, whose flowers are white and bloom in December and March, can not bear fruit and grows on the stem of the scales. The scales stem is known to be used medicinally in the genomes, pertussis, etc. However, the therapeutic effect on TRAIL-resistant cancer is not known and it was firstly described by the present inventors.

The term "extract" in the present invention refers to an extract, such as an extract obtained by extracting a daffodil, a diluted solution or concentrate of the extract, a dried product obtained by drying the extract, a controlled preparation or a purified product of the extracted solution, Lt; RTI ID = 0.0 > formulations < / RTI >

The narcissus extract of the present invention can be extracted from various organs of natural, hybrid or variant plants of the above-mentioned daffodils and extracted from plant roots, roots, stems, leaves, flowers as well as plant tissue cultures And preferably an extract of the whole plant part of the daffodil.

In the above-described narcotic extract of the present invention, the method of extracting the narcissus is not particularly limited, and may be extracted according to a method commonly used in the art. Examples of the extraction method include hydrothermal extraction, cold extraction, warm-up extraction, pressure extraction, ultrasonic extraction, filtration, and reflux extraction. These may be carried out singly or in combination with two or more methods have.

In the present invention, the kind of the extraction solvent used for extracting the spermatogen is not particularly limited, and preferably, it can be extracted using water, an organic solvent, or a mixed solvent thereof. In the case of extraction using an organic solvent, a C ₁ to C ₄ alcohol such as methanol, ethanol, isopropanol or butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, Organic solvent such as formamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof may be used, and the effective ingredient of the herbal medicine may not be destroyed or minimized, . Depending on the organic solvent to be extracted, the degree of extraction and the degree of loss of the active ingredient of the medicament may differ. Therefore, an appropriate organic solvent should be selected and used.

"Fraction" in the present invention means a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

The fractionation method for obtaining the fraction in the present invention is not particularly limited and may be carried out according to a method commonly used in the art. As a non-limiting example of the above-mentioned fractionation method, fractions can be obtained from the extract by treating a predetermined solvent with the extract obtained by extracting the daffodil.

The kind of the fraction solvent used for obtaining the fraction in the present invention is not particularly limited and any solvent known in the art can be used. Non-limiting examples of the fraction solvent include polar solvents such as water and alcohol; And non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These may be used alone or in combination of two or more. When alcohols are used in the fraction solvent, C ₁ to C ₄ alcohols can be preferably used.

The inventive extract of the present invention or its fractions is characterized in that it increases the sensitivity to TRAIL protein and thus shows a therapeutic effect on TRAIL-resistant cancer, which was first identified by the present inventors.

In the present invention, "TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) protein" is a member of cytokine protein belonging to the tumor necrosis factor (TNF) superfamily, GenBank of NCBI, and the like. 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 combined administration of the newly identified daffylate extract or its fractions of the present inventors has a characteristic that TRAIL-resistant cancer effectively exhibits the cancer cell killing effect of TRAIL.

In the present invention, "cancer" is a disease associated with the regulation of cell death, collectively referred to as a disease caused by excessive proliferation of cells when a normal balance of apoptosis is broken. These abnormal hyperproliferative cells invade surrounding tissues and organs in some cases to form masses (masses), and they may cause destruction or deformation of normal structures in the body. Such conditions are collectively referred to as cancer.

In general, a tumor refers to a mass abnormally grown by autonomous overgrowth of the body tissue. The tumor can be divided into benign tumor and malignant tumor. Malignant tumors have a much faster growth rate than benign tumors, and they invade the surrounding tissues, causing metastasis and endangering life. These malignant tumors are commonly referred to as 'cancer'.

In the present invention, the kind of the cancer is not particularly limited, but means a TRAIL-resistant cancer for the purpose of the present invention. Non-limiting examples of such cancers include cerebrospinal tumors, head and neck cancer, lung cancer, breast cancer, thymoma, mesothelioma, esophageal cancer, cervical cancer, colon cancer, liver cancer, gastric cancer, pancreatic cancer, biliary cancer, kidney cancer, bladder cancer, prostate cancer, , Ovarian cancer, cervical cancer, endometrial cancer, colon cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma and the like.

In the present invention, "TRAIL-resistant cancer" means a cancer that is not effectively prevented, ameliorated or treated by treatment with TRAIL protein. More specifically, TRAIL protein receptor is expressed at a low level on the cancer cell surface , Which are not susceptible to TRAIL protein from the beginning, have anticancer effects due to treatment with TRAIL protein as well as all types of cancer in which apoptosis mechanism induced by TRAIL protein is not effective, It includes all of the cancer cells whose TRAIL protein is gradually reduced or the effect is lost by protein treatment.

In the present invention, the TRAIL-resistant cancer includes all kinds of cancer without any particular limitation as long as it is a cancer showing resistance to TRAIL. Non-limiting examples of such TRAIL-resistant cancers include lung cancer, gastric cancer, liver cancer, breast cancer, prostate cancer, colon cancer, kidney cancer, skin cancer, glioma cancer, thyroid cancer, blood cancer and the like.

In one embodiment of the present invention, the therapeutic effect of a representative TRAIL-resistant cancer against the lung cancer cell line A549 was confirmed (Example 2), and when such a daffodil extract or its fractions of the present invention were administered in combination with TRAIL, TRAIL- , It is obvious that the same treatment effect is also applied to other types of TRAIL-resistant cancer cells.

In the present invention, "prevention" means any action to inhibit or delay the onset of cancer by administering the pharmaceutical composition of the present invention to a subject, and "treatment" means that the pharmaceutical composition of the present invention is administered It means any act that causes the symptom of cancer to be improved or benefited.

When the pharmaceutical composition of the present invention is treated with TRAIL-resistant cancer cells, the expression of the TRAIL protein receptor is increased in cancer cells by the action of the above-described Daffodil extract or its fraction, and the expression of the TRAIL protein receptor is increased Treatment of TRAIL protein with increased susceptibility to TRAIL can effectively prevent, ameliorate, or treat cancer that has previously been resistant to TRAIL.

In one embodiment of the present invention, the truncation of the PARP protein and the BID protein was significantly increased when the present invention was applied to the lung cancer cell line A549, which is a typical TRAIL-resistant cancer, and the TRAIL- As a result of the staining, it was confirmed that the proportion of cells showing electrophoresis and late apoptosis cell death was greatly increased (Example 4). This suggests that the increase of TRAIL protein sensitivity of A549 lung cancer cell line by the combined use of Danish seed extract and TRAIL is due to an increase of extrinsic apoptosis cell death.

In the pharmaceutical composition of the present invention, the above narcotic extract or fraction thereof may be contained in an amount of preferably from more than 0 to 50 μg / ml based on the total volume of the pharmaceutical composition, more preferably from 1 μg / Ml < / RTI > to 30 g / ml. Within the above range, the anticancer effect, including the effect of increasing the expression of the TRAIL protein receptor depending on the extract of the narcissus or its fractions, can be economically achieved by using the extract of the narcissus extract or its fractions in an appropriate amount to achieve the object of the present invention There is an advantage that it is more suitable for

In the pharmaceutical composition of the present invention, the TRAIL protein may be contained in an amount of preferably more than 0 to 200 ng / ml, more preferably 1 to 100 ng / ml, based on the total volume of the pharmaceutical composition, ng / ml. < / RTI > Within the above-mentioned range, the TRAIL protein is used in an appropriate amount, which is economical, and an anticancer effect including induction of apoptosis according to the TRAIL protein is sufficiently exhibited, which is more suitable for achieving the object of the present invention .

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier as well as the above-described Draized extract or its fractions and the TRAIL protein.

In the present invention, the above-mentioned "pharmaceutically acceptable" means that it is commonly used in the pharmaceutical field, which does not disturb the biological activity and properties of the compound administered, without irritating the organism upon administration thereof.

In the present invention, the type of the carrier is not particularly limited and any carrier conventionally used in the art can be used. Examples of the carrier include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, . These may be used alone or in combination of two or more.

In addition, the pharmaceutical composition of the present invention may be supplemented with other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, if necessary, and may be used as fillers, extenders, wetting agents, disintegrants, An activator, a binder, a lubricant, or the like may be additionally used.

 The pharmaceutical composition of the present invention may be formulated into various formulations suitable for oral administration or parenteral administration.

Non-limiting examples of such oral dosage forms include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs .

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; Excipients such as dicalcium phosphate and the like; Disintegrating agents such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax may be used, and sweeteners, fragrances, syrups and the like may also be used. . Furthermore, in the case of capsules, in addition to the above-mentioned substances, liquid carriers such as fatty oils can be further used.

Non-limiting examples of the parenteral preparation include injections, suppositories, respiratory inhalation powders, aerosol preparations for spraying, ointments, powder for application, oils, creams and the like.

In order to formulate the pharmaceutical composition of the present invention for parenteral administration, a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, a lyophilized preparation, an external agent, and the like may be used. Examples of the non-aqueous solution and the suspension include propylene glycol, Polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.

More specifically, when the pharmaceutical composition of the present invention is formulated into an injection, the composition of the present invention is mixed with water or a stabilizer or a buffer in water to prepare a solution or suspension, which is then mixed with an ampoule or a vial, And the like. When the pharmaceutical composition of the present invention is formulated into an aerosol formulation, a propellant or the like may be added together with the additive such that the water-dispersed concentrate or the wet powder is dispersed.

When the pharmaceutical composition of the present invention is formulated into an ointment, cream, or the like, it may be an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, a polyethylene glycol, a silicone, a bentonite, Zinc or the like as a carrier.

The pharmaceutically effective amount and the effective dose of the pharmaceutical composition of the present invention may be varied depending on the formulation method, administration method, administration time and / or route of administration of the pharmaceutical composition. The type and severity of the response, the type of subject being treated, the age, body weight, general health status, severity or severity of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously with the subject, , And the like, and those skilled in the art will readily determine and prescribe dosages that are effective for the desired treatment.

The dosage for the more preferable effect of the pharmaceutical composition of the present invention may preferably be 10 mg / kg to 1,000 mg / kg per day, more preferably 50 mg / kg to 500 mg / kg per day. Administration of the pharmaceutical composition of the present invention can be administered once a day, or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The route of administration and the mode of administration of the pharmaceutical composition of the present invention may be independent of each other and are not particularly limited in the method and may be arbitrarily administered and administered as long as the pharmaceutical composition can reach the desired site It is possible to follow the method. The pharmaceutical composition may be administered orally or parenterally.

The parenteral administration may be, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, and a method of applying, spraying or inhalation the composition to a diseased site may also be used But are not limited to.

The pharmaceutical composition of the present invention may preferably be administered orally or by injection, more preferably orally.

According to another aspect of the present invention, there is provided a method for preventing or treating TRAIL-resistant cancer by administering the pharmaceutical composition of the present invention to an individual.

The term "individual" as used herein refers to all animals, including mammals including rats, livestock, humans, and the like.

In the method for preventing or treating TRAIL-resistant cancer of the present invention, the dose, route of administration, administration mode, etc. of the pharmaceutical composition are the same as those described above in connection with the pharmaceutical composition of the present invention.

According to still another aspect of the present invention, there is provided a health functional food composition for preventing or ameliorating a TRAIL-resistant cancer containing the extract of the daffodil or a fraction thereof and TRAIL protein as an active ingredient.

In the present invention, "improvement" means all actions that at least reduce the degree of symptom associated with the condition being treated, for example.

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.

According to another aspect of the present invention, there is provided a feed additive or a drinking water additive for preventing or improving a TRAIL-resistant cancer containing the extract of the narcissus or a fraction thereof and TRAIL protein as an active ingredient.

The feed additive or drinkable water additive of the present invention may be prepared by separately preparing the above-described daffodil extract or its fractions, and a composition containing the TRAIL protein in the form of a feed additive or a drinkable water additive, mixing the mixture with a feed or drinking water, Fractions thereof, and compositions containing the TRAIL protein can be used directly in the production of feed or drinking water.

The feed additive or drinking water additive of the present invention may be in a liquid or dry state, preferably in the form of a dried powder. The drying method for preparing the feed additive or the additive for drinking water of the present invention in the form of a dried powder is not particularly limited and a method commonly used in the art can be used. Non-limiting examples of the drying method include air drying, natural drying, spray drying, and freeze drying. These may be used alone or in a manner that uses two or more methods together.

The feed additive or the drinking water additive of the present invention may further include other additives as required. Non-limiting examples of the above-mentioned usable additives include binders, emulsifiers, preservatives and the like which are added to prevent deterioration of feed or drinking water quality; A non-protein nitrogenous compound, a silicate, a buffer, a coloring agent, an extracting agent or an oligosaccharide to be added for the purpose of increasing the efficiency of feed or drinking water, In addition, a feed mixture or the like may be further included. These may be used alone or two or more of them may be added together.

According to another aspect of the present invention, there is provided an extract or fraction thereof of daffodil and a feed or drinking water for preventing or improving TRAIL-resistant cancer comprising TRAIL protein as an active ingredient.

The feed or drinking water can be prepared by adding the feed additive or drinking water additive of the present invention to feed or drinking water, or directly adding the composition containing the above-described narcotic extract or fraction thereof and TRAIL protein.

In the present invention, the type of the feed is not particularly limited, and feeds conventionally used in the art can be used. Non-limiting examples of such feeds include vegetable feeds such as cereals, muscle roots, food processing busines logistics, algae, fibers, pharmaceutical buses, oils, fats, pastes or grain by-products; Animal feeds such as proteins, inorganic substances, fats, oils, fats, oils, monocellular proteins, animal plankton or foods. These may be used alone or in combination of two or more.

In the present invention, the type of the drinking water is not particularly limited, and drinking water commonly used in the related art can be used.

According to another aspect of the present invention, there is provided a first composition comprising, as an active ingredient, a narcotic extract or a fraction thereof; And a second composition containing an TRAIL protein as an active ingredient. The present invention also provides a kit for preventing or treating TRAIL-resistant cancer.

The kind of the kit of the present invention is not particularly limited, and a kit of a type commonly used in the art can be used.

The kit of the present invention may be packaged in the form that the first composition and the second composition are each contained in a separate container or contained in one container divided into one or more compartments and the first composition and the second composition May be packaged in the form of a unit dose of one dose each.

The first composition and the second composition in the kit may be administered jointly or separately together at an appropriate time depending on the health condition of the subject to be administered.

According to another aspect of the present invention, there is provided a method for preventing or treating TRAIL-resistant cancer using the kit.

The method for preventing or treating cancer using the kit of the present invention includes administering the first composition and the second composition contained in the kit to a subject.

In the method for preventing or treating cancer using the kit of the present invention, the dose, administration route, mode of administration, etc. of the first composition and the second composition of the kit are the same as those of the pharmaceutical composition of the present invention The same as described above.

In the method for preventing or treating cancer using the kit of the present invention, the first composition and the second composition may be administered independently at each administration time, administration frequency, administration route, administration mode, and the like. Specifically, the first composition and the second composition may be administered by the same or different dosing regimes, either simultaneously or at the same time, with the same or different dosing frequency, through the same or different administration routes.

In the present invention, the first composition may preferably be orally administered, and the second composition may preferably be administered orally or by injection.

In the present invention, the first composition and the second composition are preferably administered in such a manner that the first composition and the second composition are administered substantially simultaneously.

In the present invention, the above narcotic extract or its fractions are the same as those described above with respect to the narcissus extract or its fractions in the pharmaceutical composition of the present invention.

In the present invention, treating the composition can be carried out by administering the composition to a separate cell or an individual. Administration of the composition to the isolated cells may include adding the composition to the culture medium of the cells or administering the composition to the cells by injection. The dose, route of administration, mode of administration and the like for administering the composition to the subject are the same as those described in connection with the pharmaceutical composition of the present invention.

Since the Daffodil extract of the present invention or its fractions has an effect of increasing the sensitivity of TRAIL-resistant cancer cells to cytotoxicity of TRAIL protein and inducing apoptosis of cancer cells, it is possible to effectively prevent, ameliorate, Or treatment.

In addition, the above-described daffodil extract or fraction thereof of the present invention is derived from a natural product and has an advantage that it can be used stably without causing severe irritation to the normal cells or causing harmful action.

1 is a graph showing the relative cell viability of TRAIL-resistant A549 lung cancer cell line and TRAIL-sensitive H460 lung cancer cell line, MCF-7 breast cancer cell line, and PC-3 prostate cancer cell line treated with TRAIL, Fig.
FIG. 2 is a graph showing the morphology of cells in a microscope after 48 hours from the treatment with Bacillus thuringiensis extract (B65) on an A549 lung cancer cell line according to an embodiment of the present invention.
FIG. 3 is a graph showing that the A549 lung cancer cell line was treated with the TRAIL protein and the Bacillus thuringiensis extract (B65) and other plant extracts in combination with the TRAIL protein, and the PARP protein Was observed with a Western blot.
FIG. 4 is a graph showing the degree of cell death induced by apoptosis in A549 lung cancer cell lines after 24 hours of treatment with A549 lung cancer cell line (B65) and TRAIL protein alone or in combination, according to an embodiment of the present invention will be.
FIG. 5 is a Western blot image showing the concentration-dependent cleavage phenomenon of BID protein and PARP protein, which are markers of extrinsic apoptosis cell death by the dandelion outpost extract (B65), according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention.

Manufacturing example  1: hydroxide Outpost  Preparation of extract

The dernication outpost was dried and pulverized, and extracted with 99.9% methanol at room temperature. The extraction was carried out by changing the solvent every time, and was repeated three times for 48 hours at a time. After the extraction, the extract was concentrated under reduced pressure to remove the oil component in the upper layer, and then the extract of the lower layer was dried and homogenized to obtain an extract of the hydroxylated precursor.

Example  1: A549's TRAIL  Identify resistance to proteins

In order to confirm TRAIL resistance of A549 human lung cancer cell line which is known to be resistant to TRAIL protein, the following experiment was conducted.

The cells were plated in 100-well RPMI (Roswell Park Memorial Institute medium) + 10 < th > wells on a 96-well plate at a dose of 5,000 per A549 and H460 human lung cancer cell line, MCF-7 breast cancer cell line, ML, 50 ng / mL, 25 ng / mL, and 50 ng / mL, respectively, in a final FBS (Fetal Bovine Serum) culture medium for 24 hours and TRAIL (Invitrogen, USA) / mL and 100 ng / mL, respectively. After 48 hours from the treatment, the cell viability in each well was measured using Ez-Cytox solution (Daeil Lab Service, Korea). The relative survival rate was calculated as the relative survival rate to the mean value of negative control (PBS) (Fig. 1).

As shown in FIG. 1, A549 cells showed cell viability of 85% or more even after treatment with 100 ng / mL of TRAIL protein for 48 hours, while H460, another lung cancer-derived cell line, or a cell line derived from another tissue , The cytotoxicity of TRAIL treatment was dependent on the concentration of TRAIL in MCF-7 (breast cancer) and PC-3 (prostate cancer). In particular, treatment with TRAIL protein at concentrations higher than 25 ng / mL showed prominent cytotoxicity .

Thus, it was confirmed that the A549 lung cancer cell line was resistant to TRAIL among the cancer cell lines.

Example  2: Daffodil from A549 lung cancer cell line outpost  Depending on the treatment of the extract Cell death  increase

In order to confirm that the cell death was increased when TRAIL-resistant A549 cells were treated with TRAIL, the 5,500 A549 lung cancer cell lines per well were inoculated into 100-well RPMI + 10 % FBS culture medium for 24 hours, and the medium was treated with TRAIL or negative control bovine serum albumin (BSA) to 100 ng / mL. At the same time, the dandelion seed extracts were combined with 100 ㎍ / mL. Forty-eight hours after the treatment, morphological changes of the cells in each well were observed with an inverted microscope.

As shown in FIG. 2, compared with the control group in which the Danish seed extract was treated with BSA, A549 cells treated with TRAIL protein showed not only slowed cell growth but also apoptotic cell death phenomenon in which the cells contracted as a whole. This suggests that the extracts of dandelion seedlings increase the sensitivity to TRAIL in A549 cells.

Example  3: Daffodil Outpost  The extract and TRAIL A549 < / RTI > Within the cell of Apoptosis Cell death  Increase observation

Example  3-1: Daffodil Outpost  Extract and TRAIL  By combination treatment Apoptosis Cell death  Observation of related protein expression

In Example 2, when the dandruff extract was treated with TRAIL protein, the apoptotic cell death was increased. In order to confirm whether the apoptotic cell death was increased in the cells, Western blot analysis was performed.

Specifically, A549 cells were cultured for 24 hours in a 60 mm culture dish, and 100 μg / mL of Drosophila extract (B65) or a negative control DMSO was treated with BSA or TRAIL 100 ng / mL for 24 hours. Then, the intracellular protein was separated using RIPA buffer and electrophoresis of 10 의 protein. The protein separated from the PAGE gel was transferred to a nitrocellulose membrane, and poly (ADP ribose polymerase) and beta - actin protein were detected as specific antibodies, respectively. Rabbit-HRP and anti-goat-HRP antibodies used as secondary antibodies in Santa Cruz Biotechnology, Inc., were used in the Western blot experiments, as well as the PARP and primary antibodies used in Cell Signaling Technologies, And purchased from Biorand Company.

As a result, as shown in FIG. 3, the extract of Drug X1-X4 did not significantly increase the cleavage of PARP protein as compared with the vehicle + TRAIL control, whereas the extract of Bacillus thuringiensis extract (B65) , The cleavage of PARP protein was significantly increased.

Example  3-2: Daffodil outpost  The extract and TRAIL In combination treatment Cell death Apophoto Determine if it is induced by cis

In order to examine whether apoptotic cell death was accompanied by apoptotic cell death, the cells were stained with Annexin-FITC and analyzed by FACS.

Specifically, the A549 lung cancer cell line was grown in a 6-well culture dish for 24 hours, and then treated with Drosophila extract (50 ㎍ / mL) and TRAIL (100 ng / mL) for 24 hours. DMSO and BSA proteins were used as negative controls for the dsRNA extract and TRAIL protein, respectively. Cells were stained with the Annexin V-FITC apoptosis detection kit supplied by BD Bioscience after the combination of the dendrillous outpost extract and TRAIL. Cells stained with Annexi V-FITC (FITC) and propidium iodine (PI) were analyzed by FACS analysis within 1 hour after staining. Cells were classified according to the following Table 1, respectively, for normal cells, electrophoretic cells, late apoptotic cells, and necrosis-induced cell death.

Cell division by FITC / PI dye combination Viable cells (V) Electrical apoptosis cells (EA) Late apoptotic cells (LA) Necrosis cells (N) FITC - + + - PI - - + +

As a result, the ratio of cell apoptotic cell death and post apoptotic apoptotic cell death was significantly increased in the case of treatment with Bacillus subtilis extract (B65) or TRAIL alone (B65 + TRAIL), respectively. Therefore, it can be confirmed that the extract of the dandelion seed extract increases apoptotic cell death by TRAIL in A549 cells (Table 1, Fig. 4).

Example  3-3: Daffodil Outpost  Extract and TRAIL  By combination treatment extrinsic Apoptosis Cell death  Observation of related protein expression

Cell death by DR4 / DR5, a cell membrane receptor of TRAIL protein and TRAIL protein, is known to follow extrinsic apoptosis signaling pathways. Specifically, when TRAIL binds to DR4 / DR5, the activity of caspase-8 is increased and the BID protein is cleaved by activated caspase-8. Therefore, it was confirmed whether the expression pattern of extrinsic apoptosis cell death related protein was changed by Drosophila extract.

Specifically, the A549 cells were cultured in a 60 mm culture dish for 24 hours, and then the dendritic cell extract (12.5-100 ㎍ / mL) and 100 ng / mL of TRAIL were used in combination. After the treatment for 24 hours, proteins in the cells were separated using RIPA buffer, and Western blot was performed as in Example 3-1. The primary antibody of BID used in Western blot was purchased from CellSignaling Technologies.

As a result, the truncated BID protein was not detected when TRAIL alone was treated without the dernicidal extract (B65). However, in the case of the combination of the Doxanthorrhizae extract and the TRAIL protein, the BID protein cleavage is increased in a concentration-dependent manner by the dandruff extract (long when the exposure is prolonged), and the amount of intact BID protein is relatively decreased (Short exposure, short exposure).

The increase in TRAIL protein sensitivity of the A549 lung cancer cell line by the dandelion outpost extract according to Example 3 is due to an increase in the extrinsic apoptosis cell death due to the danshimae outpost extract.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. More variations are possible within a range that does not. Accordingly, the present invention may be embodied in other ways than those specifically described and illustrated herein, and it may be understood by those skilled in the art.

Claims (8)

A pharmaceutical composition for preventing or treating TRAIL-resistant lung cancer, which comprises an extract of Narcissus tazetta var. Chinensis or a fraction thereof, and a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein as an active ingredient.
delete 2. The composition of claim 1, wherein the narcissus is a narcotic herb.
A method for preventing or treating cancer, comprising administering the composition of claim 1 or 3 to a subject other than a human.
A health functional food composition for preventing or ameliorating TRAIL-resistant lung cancer, comprising a daffodil extract or a fraction thereof and TRAIL protein as an active ingredient.
A drinking water for the prevention or improvement of TRAIL-resistant lung cancer, containing the extract of the daffodil or its fraction, and TRAIL protein as an active ingredient.
6. The composition of claim 5, wherein the narcissus is a narcotic herb.
A feed for preventing or improving TRAIL-resistant lung cancer, comprising a daffodil extract or a fraction thereof, and TRAIL protein as an active ingredient.

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