KR102450560B1 - Composition for preventing and treating a blood cancer comprising extracts of Draconis sanguis - Google Patents

Abstract

The present invention relates to a composition for preventing, treating or improving blood cancer, comprising an extract of Draconis sanguis, wherein the blood extract of the present invention induces a endoplasmic reticulum-stress response in blood cancer cells to prevent apoptosis By inducing it, it inhibits the proliferation of cancer cells and causes their death, so the composition comprising the blood extract can be used for preventing, treating or improving blood cancer.

Description

Composition for preventing or treating a blood cancer comprising extracts of Draconis sanguis as an active ingredient

The present invention relates to a composition for preventing, treating or improving blood cancer, comprising an extract of Draconis sanguis.

ER stress (endoplasmic reticulum stress; ER stress) refers to the failure of the ER function due to the influx of immature proteins beyond the capacity of the ER to the ER or the depletion of calcium in the ER by the physiological or pathological environment. Specifically, in the ER, misfolding proteins and overexpressed proteins bind to the chaperone Bip/Grp78 (ER transmembrans receptor protein) in the lumen of the ER, thereby initiating the Unfoded Protein Response (UPR) mechanism. In normal conditions, it is not active, but is separated from the receptor by binding to misfolded protein and overexpressed protein, and then PERK (protein kinase RNA-like endoplasmic reticulum kinase) is activated by autophosphorylation in the cytoplasm, and protein synthesis In order to weaken the mRNA, phosphorylation is regulated by negatively regulating the elf2 factor, which is an initiation factor for mRNA binding (negative feedbeck). In this process, the G1 phase of the cell cycle is suppressed and the most important CHOP (CCAAT/-enhancer-binding protein homologuous protein; C/EBP homologous protein) is expressed in ER stress. Subsequently, cleavage of PARP related to apoptosis is induced by mitochodrial damage. The most important CHOP is to generate reactive oxygen species (ROS).

On the other hand, the smallest unit constituting the human body is called a cell, and normal cells divide and grow, die and disappear due to intracellular regulatory functions, maintaining the balance of cell numbers. When cells are damaged for some reason, they function as normal cells through treatment, or they die by themselves if there is no recovery. However, when a cell's gene is changed due to various reasons, the cell changes abnormally and matures incompletely, and the cell cycle is not regulated, so cell division continues. This is defined as cancer.

Cancer is broadly classified into blood cancer and solid cancer, and occurs in almost all parts of the body, including lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer. According to the data of the Central Cancer Registry published in 2016, there were 217,057 cancer cases in Korea in 2014. Among them, multiple myeloma (C90) was 1,396 cases, accounting for 0.6% of all cancer cases, and it is difficult to cure when it is diagnosed. one of the diseases

Multiple myeloma is a representative blood tumor along with leukemia and lymphoma, and is a blood cancer in which plasma cells, a mature form of B lymphocytes, proliferate. On average, it occurs in elderly people over 65 years of age, and the incidence rate is relatively low in Asians. However, the number of patients with multiple myeloma continues to increase due to pollution, increased exposure to environmental hormones, and an aging population due to rapid industrialization. In the past 20 years, the incidence of other carcinomas has only increased five-fold, but in the case of multiple myeloma, the incidence has increased more than 30-fold.

The treatment of multiple myeloma is standard chemotherapy (MP therapy) using melphalan and prednisone, high-dose chemotherapy and hematopoietic stem cell transplantation applied to patients under the age of 65, and refractory cancer or recurrent myeloma that does not respond to conventional treatment. There are targeted therapies for However, due to the occurrence of resistant and refractory cancers to currently used drugs, the mortality rate from multiple myeloma is very high. In particular, bortezomib, a targeted treatment that has recently obtained FDA approval as a second-line drug, has been reported to be highly effective in refractory and recurrent cancers, but the overall response rate is 35% and the complete remission rate is insignificant at 10%. Drug-resistant cancer has been reported. Therefore, there is a need to develop a therapeutic regimen that can dramatically increase the survival rate of patients with multiple myeloma by effectively treating refractory or recurrent cancer of multiple myeloma.

On the other hand, blood blood (Draconis Sanguis) is a drug widely used in China, and most studies have been conducted on trauma, inflammation, gynecopathy, and allergic dermatitis. nothing has happened

Therefore, the present inventors were trying to find a natural product with few side effects that can prevent and treat blood cancers such as multiple myeloma and leukemia, and it was confirmed that blood flow in blood cancer cells induces endoplasmic reticulum-stress and effectively apoptosis. and completed the present invention.

Korean Patent No. 10-2050639

An object of the present invention is to provide a pharmaceutical composition for preventing or treating blood cancer.

Another object of the present invention is to provide an anticancer adjuvant for hematologic cancer.

Another object of the present invention is to provide a food composition for preventing or improving blood cancer.

Another object of the present invention is to provide a method for preventing or treating hematologic cancer.

Another object of the present invention is to provide a method for increasing PARP cleavage and CHOP expression in vitro.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating blood cancer comprising an extract of Draconis sanguis as an active ingredient.

In addition, the present invention provides an anti-cancer adjuvant for blood cancer comprising a blood extract as an active ingredient.

Furthermore, the present invention provides a food composition for the prevention or improvement of blood cancer, comprising the blood extract as an active ingredient.

Furthermore, the present invention provides a method for preventing or treating blood cancer by administering a pharmaceutically effective amount of a blood extract to a subject other than a human in need thereof.

Furthermore, the present invention provides a method of increasing PARP cleavage and CHOP expression by treating blood cancer cells in vitro with blood cancer cells.

Draconis sanguis extract of the present invention induces endoplasmic reticulum-stress response in blood cancer cells and induces apoptosis, thereby inhibiting the proliferation of cancer cells and causing their death. It can be used for preventing, treating or improving cancer.

1 is a view confirming the cell viability of the blood cancer cell lines U937 and THP-1 by the treatment of blood extract.
2 is a diagram confirming the expression level of apoptosis and endoplasmic reticulum-stress-related proteins in the blood cancer cell lines U937 and THP-1 by the treatment with blood extract.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for prevention or treatment of hematologic cancer

The present invention relates to a pharmaceutical composition for preventing or treating blood cancer comprising an extract of Draconis sanguis as an active ingredient.

In the present invention, the 'blood blood (血竭, Draconis Sanguis)' refers to a mass made by heat-pressing the resin exuded from the fruit of Kiringal (麒麟竭) Daemonorops draco Blume .

As used herein, the term "extract" refers to a preparation obtained by squeezing a crude drug with an appropriate leachate and evaporating the leachate, but is not limited thereto, but includes an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, It may be a dried product obtained by drying the extract, a crude product thereof, or a purified product. The extract may be prepared using a general extraction method, separation and purification method known in the art. The extraction method is not limited thereto, but preferably, methods such as hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction may be used.

In the present invention, the blood extract can be prepared by fractionation by adding a fractionation solvent to the extract prepared by extraction with an extraction solvent or extraction with an extraction solvent. The extraction solvent is not limited thereto, but water, an organic solvent or a mixture thereof may be used, and the organic solvent is an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dichloro. A non-polar solvent of methane or a mixed solvent thereof may be used. In addition, preferably water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof may be used, and more preferably ethanol may be used.

In one embodiment of the present invention, extraction was performed using 100% ethanol as the solvent and then concentrated under reduced pressure to prepare a blood extract. In this case, 100% ethanol in an amount corresponding to 8 to 12 times the volume based on the weight of the blood powder may be mixed and extracted for 2 hours or more, preferably 2 to 10 hours, but is not limited thereto.

In one embodiment, the cancer may be any one or more selected from the group consisting of leukemia, multiple myeloma, lymphoma, gastric cancer, lung cancer, liver cancer, breast cancer and prostate cancer, preferably multiple myeloma or leukemia. and multiple myeloma is more preferred.

In one embodiment, the effective mechanism of the prevention or treatment of hematologic cancer may be achieved by apoptosis due to endoplasmic reticulum (ER) stress of cancer cells.

As used herein, the term "apoptosis" is also referred to as apoptosis or apoptosis, and is a kind of programmed cell death (PCD). It is a mechanism that allows cells to self-destruct and die to maintain overall body health.

According to an embodiment of the present invention, the extract may induce apoptosis due to endoplasmic reticulum (ER) stress of blood cancer.

According to an embodiment of the present invention, the extract increases PARP (Poly ADP ribose polymerase) cleavage and increases apoptosis and endoplasmic reticulum-stress by increasing CHOP (C/EBP homologous protein) expression.

According to an embodiment of the present invention, the composition may inhibit or reduce the survival rate of multiple myeloma cells or leukemia cells.

In one embodiment of the present invention, it was confirmed that the blood extract of the present invention increases PARP cleavage and increases GADD153/CHOP expression in U937 and THP-1 cells, which are multiple myeloma cell lines. It was confirmed that cell death can be effectively induced by increasing endoplasmic reticulum-stress (see Experimental Examples 1 to 3).

In the present invention, the term "prevention" refers to any action that suppresses or delays the occurrence, spread, and recurrence of cancer by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to any action that is performed by administration of the pharmaceutical composition. It refers to any action that improves or beneficially changes the symptoms of a suspected or affected individual.

As used herein, the term “treatment” refers to all methods of improving or beneficially changing the apoptosis of cancer cells or symptoms of cancer by administering the composition comprising blood flow of the present invention. Those of ordinary skill in the art to which the present invention pertains, with reference to the data presented by the Korean Medical Association, etc., know the exact criteria of the disease for which the composition of the present application is effective, and can determine the degree of improvement, improvement and treatment will be.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention means an amount of a pharmaceutically acceptable salt of the blood extract effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention. The amount may vary depending on several factors, for example, the method of administration, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations when determining the effective amount are described, for example, in Effect of Sanguis Draconis on Perforator Flap Survival in Rats, Yang Zhang et al., Molecules, 2016).

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, type of cancer, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological agents. As used herein, the term “pharmaceutically acceptable” refers to exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compound described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate. , lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol and talc and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The pharmaceutical composition of the present invention may further include a known anticancer agent in addition to the blood extract as an active ingredient, and may be used in combination with other known treatments for the treatment of these diseases. Other treatments include, but are not limited to, chemotherapy, radiation therapy, hormone therapy, bone marrow transplantation, stem-cell replacement therapy, other biological therapies, immunotherapy, and the like.

Examples of anticancer agents that may be included in the pharmaceutical composition of the present invention include mechloethamine, chlorambucil, phenylalanine, mustard, cyclophospha as DNA alkylating agents. Cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin ( cisplatin) and carboplatin; As anti-cancer antibiotics, dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin, idarubicin, mitoxantrone, plicama plicamycin, mitomycin C and bleomycin; and plant alkaloids vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan and iridotecan, and the like, but are not limited thereto.

How to prevent or treat blood cancer

In one aspect, the present invention relates to a method for preventing or treating blood cancer, comprising administering the blood extract to a subject in need thereof.

As used herein, the term "individual" means a monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or It means all animals, including guinea pigs, and can effectively prevent or treat the above diseases by administering the pharmaceutical composition of the present invention to an individual.

In one aspect, the present invention relates to a method for preventing or treating blood cancer by administering a pharmaceutically effective amount of a blood extract to a subject other than a human in need thereof.

In addition, the present invention provides a method for reducing the survival rate of blood cancer cells by administering a pharmaceutically effective amount of the blood extract to a subject other than a human.

In addition, the present invention provides a method for reducing the survival rate of blood cancer cells by administering a pharmaceutically effective amount of the blood extract to a subject other than a human.

The pharmaceutical composition of the present invention may be administered in parallel with a conventional therapeutic agent.

As used herein, the term "administration" means providing a predetermined substance to a patient (or subject) by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intravenous, subcutaneous, Intraperitoneal or topical injection formulation) or oral administration, the dosage varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. . Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives etc. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. The pharmaceutical composition of the present invention may be administered by any device capable of transporting an active substance to a target cell. Preferred administration methods and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. For injection, aqueous solvents such as physiological saline solution and Ringel's solution, vegetable oil, higher fatty acid esters (eg, ethyl oleate), and non-aqueous solvents such as alcohols (eg, ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) Stabilizers for preventing deterioration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, to inhibit microbial growth Pharmaceutical carriers such as preservatives (eg, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

Anticancer adjuvant for blood cancer

In one aspect, the present invention relates to an anticancer adjuvant for hematologic cancer comprising a blood extract as an active ingredient.

By administering the blood extract of the present invention together with a chemical anti-cancer drug (anti-cancer agent), it is possible to increase the cancer treatment effect of the conventional anti-cancer agent through the apoptosis effect of cancer cells. The combined administration may be performed simultaneously or sequentially with the anticancer agent.

Food composition for prevention or improvement of blood cancer

In one aspect, the present invention relates to a food composition for preventing or ameliorating cancer containing a blood extract.

When the composition of the present invention is used as a food composition, the blood extract may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. In addition to the active ingredient, the composition may include a food additive that is pharmaceutically acceptable, and the mixing amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).

As used in the present invention, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful for the human body. Here, the term 'functionality' refers to obtaining useful effects for health purposes such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the ordinary technical field, and in the production, it can be prepared by adding raw materials and components that are conventionally added in the conventional technical field. In addition, the dosage form of the health functional food may also be manufactured without limitation as long as it is a dosage form recognized as a health functional food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long period of time using food as a raw material, and it is excellent in portability, and the present invention health functional food can be taken as an adjuvant to enhance the effect of anticancer drugs.

In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, the composition comprising the blood extract of the present invention as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in health functional foods according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract according to the present invention as a main component to juice, tea, jelly, juice, and the like.

Methods to increase PARP cleavage and expression of CHOP in vitro

In one aspect, the present invention provides a method for increasing PARP cleavage and CHOP expression by treating blood cancer cells with a blood cancer cell in vitro .

The blood extract of the present invention can induce apoptosis through endoplasmic reticulum stress in blood cancer cells by increasing PARP cleavage and GADD153/CHOP expression when treated in a blood cancer cell line in vitro .

The hematological cancer cell line may be a multiple myeloma or leukemia cell line, U937 or THP-1 cell line.

Since the blood extract of the present invention is made from a natural plant as a raw material, even when used as a pharmaceutical composition or a food composition, side effects may be less than that of a general synthetic compound.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

<Example 1> Preparation of blood extract

After extracting blood blood with 100% ethanol, it was concentrated under reduced pressure to prepare an ethanol extract of blood blood.

Specifically, blood blood was purchased at Yakwon Oriental Pharmacopoeia, and 2000 mL of 100% ethanol was mixed with 200 g of blood blood powder, followed by solvent extraction for 3 hours. The extract was filtered and then concentrated under reduced pressure to prepare an ethanol extract from blood.

<Experimental Example 1> Preparation of an ex vivo cell line model

After dispensing 50 μl of the hematological cancer cell line U937 cell line, THP-1 (Korea Cell Line Bank) to 1×10 ⁴ cells/well in a 96 well-plate, 10% inactivated fetal bovine serum and 1% penicillin-streptomycin were added. In one RPMI medium, cultured in a 5% CO2 incubator at 37°C (MCO-15AC, Sanyo, Osaka, Japan). Cells were cultured in RPMI medium supplemented with 10% inactivated fetal bovine serum and 1% penicillin-streptomycin at 37° C. in a 5% CO ₂ incubator (MCO-15AC, Sanyo, Osaka, Japan).

<Experimental Example 2> Confirmation of the apoptosis effect of the blood extract on blood cancer

In order to confirm the anticancer activity of the blood cancer extract against blood cancer, the blood cancer cell line U937 and THP-1 cells were treated with the blood cancer extract of the present invention and cytotoxicity was confirmed.

Cell lines U937 and THP-1 cells cultured in Experimental Example 1 were treated with the blood extract prepared in Example 1 at a concentration of 0, 25, 50, 100, 200, and 400 μg/ml by 50 μl, respectively. The hematological cancer cell lines U937 and THP-1 were cytotoxic to a 96-well-plate and dispensed so as to become 1×10 ⁴ cells/well. After incubating the U937 and THP-1 cells treated with the blood extract for 24 hours, the cells were treated with 10 μl of EZ-Cytox reagent (DoGen) and incubated in the dark for 30 minutes to 4 hours, followed by microplate, rate reader Cell viability was confirmed by measuring absorbance at 450 nm using (Bio-rad Model 680).

As a result, as shown in FIG. 1 , the concentration-dependent toxicity of the blood extract was shown in U937 and THP-1 cells.

<Experimental Example 3> Confirmation of apoptosis and endoplasmic reticulum stress-induced effect of blood cancer cell line by treatment with blood extract

In order to confirm the effect of blood extract on apoptosis in blood cancer, it induces DNA damage with PARP (Poly ADP ribose polymerase), which is known to be cleaved during apoptosis, and induces endoplasmic reticulum stress, known as an important factor in apoptosis. Expression of C/EBP homologous protein (CHOP) was confirmed by Western blot analysis.

Specifically, in the hematological cancer cell lines U937 and THP-1 cell lines, the blood extract prepared in Example 1 was treated with 0, 15. 30 μg/ml, respectively, and incubated with a 5% CO ₂ incubator at 37° C. for 24 hours. did. Thereafter, the cells were harvested, washed with PBS, and cell lysis buffer RIPA (20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM NaEDTA, 1 mM EGTA, 1% NP-40, 1% sodium pyrpphophate, 1 mM beta-glycerophosphate) , 1 mM Na ₃ VO ₄ and 1 μg/ml leupeptin (Cell signaling)) were added and the cells were disrupted at 4° C. for 30 minutes. Cell lysate was centrifuged at 12000rpm 4℃ for 20 minutes to remove cell membrane components, and then protein was quantified using RC DC™ Protein Assay Kit II (protein assay kit) (Bio-rad, USA) by sample (blood blood extract). 0, 15, and 30 μg/ml treatment groups) were adjusted to contain the same amount of protein. A 5× loading die was put into the prepared protein sample, and the protein was denatured by heating at 100° C. for 5 minutes. Then, the sample was loaded on an 8-10% SDS-PAGE gel and electrophoresis was performed at 100 V for 2 hours and 30 minutes, and the proteins separated on the gel were transferred at 300 mA for 2 hours. After blocking the protein-transferred membrane with a TBST (tris buffered saline, pH 7.5) solution containing 5% skim milk at room temperature for 30 minutes, the primary antibody anti-PARP (Cell signaling) and anti-GADD153/CHOP (Santa cruz) was diluted at 1:1000 and 1:500, respectively, and put into the blocking membrane, and reacted overnight at 4°C so that the antibodies attach to specific positions of the protein. The next day, the membrane was washed twice with TBST, diluted with horseradish peroxidase-conjugated anti-rabbit IgG 1:10,000, reacted with the membrane at room temperature for 1 hour, and then washed twice with TBST. The washed membrane was reacted with a chemiluminescent reagent for 1 minute, and the film was exposed to light to visualize the protein band.

As a result, as shown in Figure 2, it was confirmed that the cleavage of PARP is increased depending on the concentration of the blood extract. In addition, by increasing the expression of CHOP by the blood extract, it was confirmed that the endoplasmic reticulum-stress increased, so that the blood extract could effectively induce apoptosis of blood cancer in a concentration-dependent manner.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than in the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (10)

delete delete delete delete delete delete delete delete delete A method of increasing PARP cleavage and CHOP expression by treating blood cancer cells with blood cancer cells in vitro .

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