KR101491914B1 - Pharmaceutical composition for preventing or treating cancer comprising the extract of Euonymi lignum suberalatu - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises an extract of ear drum extract as an active ingredient, and provides a pharmaceutical composition for preventing or treating cancer, which promotes aging of cancer cells to inhibit the growth of cancer cells. By promoting the aging of cancer cells, it can be useful for treating diseases such as cervical cancer, chondrosarcoma, fibrosarcoma, and colon cancer.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating cancer, which contains an extract of Echinochloa crus-galli as an active ingredient,

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, which comprises an extract of Echinochloa crus-galli as an active ingredient, and which is capable of inhibiting the growth of cancer cells by promoting cell senescence of cancer cells, ≪ / RTI >

Cell senescence is a state in which normal cells do not divide after a certain number of splits in the growth process and growth ceases. Cell senescence is called replicative senescence because normal somatic cells divide and telomeric chromosomes become shorter. In addition to the internal factors such as telomere shortening in the process of cell division, cell senescence also occurs due to various physiological and chemical stimuli such as severe inflammatory reaction, oxidative stress, DNA damage, ultraviolet radiation or irradiation, and exposure to anticancer drugs. When the cells are aged, various changes are observed in cell shape, biochemistry, molecular biology, and nuclear. When the cells are old, the cell shape becomes flattened, the size increases, and the granules increase in the cytoplasm. Biochemical and molecular biologic changes are associated with increased senescence-associated β-galactosidase (SA-β-gal) activity, increased expression of cancer suppressor genes such as p53 and p16, , Extracellular matrix proteases, cytokines, and the like. Changes in nuclei are characteristic of senescence-associated heterochromatin foci (SAHF) and DNA damage foci such as pH2AX, a DNA damage marker.

Cell senescence is known to contribute to various physiological and pathological phenomena such as tissue and individual senescence, tissue regeneration, cancer development and cancer suppression, and aging related diseases. The telomere length of leukocytes is related to diabetes, hypertension, myocardial infarction, chronic heart failure, liver cirrhosis, vascular dementia, arteriosclerosis, Alzheimer's disease, increased mortality and life expectancy at age 60 and older . In telomerase deficient mice, life span decreased after 6th generation due to heart disease, insulin resistance, stem cell dysfunction, decreased stress resistance, and increased cancer incidence. Conversely, reactivation of telomerase in telomerase deficient mice is known to improve the degenerative changes of tissue following aging. It has been reported that substances promoting telomerase activity in natural products have been discovered, increasing health life span without increasing cancer incidence in mice, and improving immune cell function in human clinical trials. It is known that selective removal of cells expressing p16 gene, which is well known to be expressed in senescent cells in animal mouse models, inhibits senescence-related diseases. Therefore, it is recognized that cell senescence plays an important role in the pathogenesis of tissue, individual senescence and aging - related diseases.

In addition, cell senescence is presented as an important internal defense mechanism to suppress cancer. Abnormal activation of various cancer genes such as Ras, Myc, and Raf and inactivation of cancer suppressor genes such as PTEN, Rb, NF1, and VHL plays an important role in the development and progression of cancer, and many cancer cells induce cell senescence And p53 / p21, Rb / p16 tumor suppressor genes. Specifically, an increase in the activity of this cancer gene promotes cell senescence, which is referred to as oncogene-induced senescence (OIS). When the activity of cancer gene is increased, DNA damage accumulates due to excessive cell proliferation. In this process, activation of cancer suppressor gene signaling pathways such as p53 and p16 leads to cell senescence and cell death, . In particular, p53, an inhibitor of cancer, plays an important role. P53 inhibits cell proliferation, induces apoptosis and cell senescence, and regulates glucose metabolism. It is known that, after inducing liver cancer in a mouse model, induction of p53 expression in cancer cells leads to suppression of cancer growth when cell senescence occurs. In addition, when cell senescence induced by Ras cancer gene is induced, immune cells have been reported to inhibit cancer development by removing aged cancer cells. These results suggest that cell senescence in the course of normal cell changes to benign cancer is an important mechanism for inhibiting the transformation of benign cancer cells into malignant tumor cells. And, in order for benign cancer cells to progress to malignant tumor cells, it is necessary to overcome the cell senescence which appears as an activity of cancer gene through any mechanism. Therefore, it is suggested that drugs capable of inducing cell senescence in cancer cells may be developed as new anticancer drugs.

Korean Patent Laid-Open No. 10-2005-0047057 discloses an extract of herbal medicine having antioxidant activity, which includes Orostachys japonicus, Sanguisorba officinalis, Lonicera japonica, Hingelia dulcis, Rhododendron brachycarpum, Winged spindle, Notoginserg Radix, Angelica dahurica, Portulaca oleracea, Rhododendron brachycarpum, Hovenia dulcis, Ulmus Macrocarpa Hance), a herbal medicine extract having ten kinds of antimicrobial activity, antitumor activity, anti-aging effect and anti-aging activity, Which is different from the present invention in its technical construction.

Accordingly, the present inventors have found out the herbal medicine extract promoting cell senescence by using human fibroblasts, and then found that the extract of the ear cane promotes cell senescence in various cancer cells such as cervical cancer cells, chondrosarcoma cells, fibrosarcoma cells, At the cellular level to complete the present invention.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, which comprises an extract of ear drum extract as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an extract of ear drum extract as an active ingredient. Specifically, the extract of the ear protector is characterized by being extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, and a mixed solvent thereof.

In detail, the extract of the senile senile worms promotes the aging of cancer cells to inhibit the growth of cancer cells. More specifically, the aging promotion of cancer cells is characterized by measuring the senescence-associated beta-galactosidase (SA-beta-gal) activity increase.

In detail, the cancer cells are preferably cervical cancer cells (Hela), chondrosarcoma cancer cells (HTB94), fibrosarcoma cancer cells (HT1080) and colorectal cancer cells (HCT116 WT, HCT116 p53 - / -, HCT116 p21 - / - But is not limited thereto.

Further, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating cancer, which can prevent or treat cervical cancer, chondrosarcoma cancer, fibrosarcoma cancer or colon cancer disease, but is not limited thereto.

The "Euonymi lignum suberalatu" of the present invention is a deciduous broad-leaved shrub with Novak vines and is often grown in mountainous areas. It reaches 3m in height and has 2 ~ 4 wings in twigs. The fruit is fruitless in October. The appearance of the wing attached to the stem is also called the 耳 箭 羽 (鬼 箭 羽), or the Shinto shrine (神 箭 木), which means an arrow that shoots a ghost. In the private sector, the stem and wing of the arrow tree were used as repellents, analgesics, and hemostats. In the oriental medicine, the arrow tree (earworm) improves the blood circulation and has the effect of eliminating the inflammation. It is used as a treatment for dermatosis by making plaster. It has the effect of stabilizing the mind and is an agent for treating mental anxiety. It has been used as a medicine to treat uterine bleeding in women because of its therapeutic effect on abdominal pain caused by it. It is known that it is effective for various cancers such as stomach cancer and esophageal cancer. It is also known that it has a therapeutic effect on asthma accompanied by hypertension, atherosclerosis and coughing sputum, which is effective for diabetes by lowering blood sugar and increasing insulin secretion.

The extract of Echinosophora koreensis is the herbal extract number CA01-018 of the Plant Extract Bank of Korea Research Institute of Bioscience and Biotechnology. It was purchased from the Plant Extract Bank of Korea Research Institute of Bioscience and Biotechnology.

In the case of the pharmaceutical composition of the present invention, the pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the extract of the ear protector. Such a pharmaceutically acceptable carrier is usually used in the preparation of a pharmaceutical, Starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxyacetate, Benzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. as an additive.

The method of administration of the pharmaceutical composition is determined depending on the severity of the cancer, and local administration is usually preferred. The dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and may be administered once to several times per day.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The pharmaceutical composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient, or may be prepared by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions, or may be in the form of elixirs, excipients, powders, granules, tablets, alerts, lotions, ointments and the like.

The present invention relates to a composition for preventing or treating cancer, which comprises an extract of ear-guinea fowl as an active ingredient, and is useful as a composition for preventing or treating cancer, which comprises an extract of the ear cane, which can inhibit the growth of cancer cells by promoting cell senescence It is about. The ear skin extract of the present invention can be used for the development of a new cancer therapeutic agent which can inhibit the growth of cancer cells by inducing cell senescence.

FIG. 1 shows the effect of CA 01-018 (Euonymi lignum suberalatu) on cell senescence and cell growth in human fibroblasts treated with adriamycin. A, SA-β-gal active staining; B, percentage of SA-β-gal positive cells; C, PARP1 / 2, caspase 3, p53 and p16 protein expression. The results were obtained after each experiment was repeated 3 times or more independently. * p < 0.05 and ** p < 0.01 vs DMSO. Cont: Control group, DMSO: dimethylsulfoxide, NAC: N-acetylcysteine, ADR: adriamycin.
Fig. 2 shows the effect of CA01-018 (Euonymi lignum suberalatu) on cytotoxicity and cell senescence in human fibroblasts. A, cytotoxicity; B, SA-β-gal active staining; C, percentage of SA-β-gal positive cells; D, PARP1 / 2, caspase 3, p53 and p16 protein expression. The results were obtained after each experiment was repeated 3 times or more independently. * p < 0.05 and ** p < 0.01 vs DMSO. Cont: control group, DMSO: dimethylsulfoxide, NAC: N-acetylcysteine.
Figure 3 shows the expression levels of the p53, p21 and p16 proteins in cancer cells. Cells were harvested and proteins were extracted. Levels of p53, p21 and p16 proteins were analyzed by Western blotting. The results were obtained after each experiment was repeated 3 times or more independently.
Figure 4 shows the effect of CA01-018 (Euonymi lignum suberalatu) on cell senescence and cell growth in human cervical cancer cells (HeLa). A, SA-β-gal active staining; B, percentage of SA-β-gal positive cells; C, cell growth; D, PARP1 / 2, caspase 3 and p53 protein expression. The results were obtained after each experiment was repeated 3 times or more independently. * p < 0.05 and ** p < 0.01 vs DMSO. Cont: control group, DMSO: dimethylsulfoxide, ADR: adriamycin.
FIG. 5 shows the effect of CA01-018 (Euonymi lignum suberalatu) on cell senescence and cell growth in human chondrosarcoma cells (HTB94). A, SA-β-gal active staining; B, percentage of SA-β-gal positive cells; C, cell growth; D, PARP1 / 2, caspase 3 and p53 protein expression. The results were obtained after each experiment was repeated 3 times or more independently. * p < 0.05 and ** p < 0.01 vs DMSO. Cont: control group, DMSO: dimethylsulfoxide, ADR: adriamycin.
FIG. 6 shows the effect of CA01-018 (Euonymi lignum suberalatu) on cell senescence and cell growth in human fibrosarcoma cells (HT1080). A, SA-β-gal active staining; B, percentage of SA-β-gal positive cells; C, cell growth; D, PARP1 / 2, caspase 3 and p53 protein expression. The results were obtained after each experiment was repeated 3 times or more independently. * p < 0.05 and ** p < 0.01 vs DMSO. Cont: control group, DMSO: dimethylsulfoxide, ADR: adriamycin.
FIG. 7 shows the effect of CA01-018 (Euonymi lignum suberalatu) on cell senescence and cell growth in human colorectal cancer cells (HCT116 WT). A, SA-β-gal active staining; B, percentage of SA-β-gal positive cells; C, cell growth; D, PARP1 / 2, caspase 3 and p53 protein expression. The results were obtained after each experiment was repeated 3 times or more independently. * p < 0.05 and ** p < 0.01 vs DMSO. Cont: control group, DMSO: dimethylsulfoxide, ADR: adriamycin.
FIG. 8 shows the effect of CA01-018 (Euonymi lignum suberalatu) on cellular senescence and cell growth in p53-deficient human colon cancer cells (HCT116 p53 - / -). A, SA-β-gal active staining; B, percentage of SA-β-gal positive cells; C, cell growth; D, PARP1 / 2, caspase 3 and p53 protein expression. The results were obtained after each experiment was repeated 3 times or more independently. * p < 0.05 and ** p < 0.01 vs DMSO. Cont: control group, DMSO: dimethylsulfoxide, ADR: adriamycin.
Figure 9 shows the effect of CA01-018 (Euonymi lignum suberalatu) on cell senescence and cell growth in p21-deficient human colon cancer cells (HCT116 p21 - / -). A, SA-β-gal active staining; B, percentage of SA-β-gal positive cells; C, cell growth; D, PARP1 / 2, caspase 3 and p53 protein expression. The results were obtained after each experiment was repeated 3 times or more independently. * p < 0.05 and ** p < 0.01 vs DMSO. Cont: control group, DMSO: dimethylsulfoxide, ADR: adriamycin.

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

< Example  1> Identification of herbal medicine extract promoting cell aging by adriamycin in human fibroblasts

1. Experimental material

Human fibroblasts were purchased from Lonza (Walkersville, MD, USA). Human cervical cancer cells, chondrosarcoma cells, and fibrosarcoma cells were purchased from Korean Cell Line Bank. HCT116 wild type (WT), HCT116 p53 - / -, and HCT116 p21 - / - cells were sold by Dr. Vogelstein from Johns Hopkins Medical School. Dubeccos-Modified Eagle's medium (DMEM), fetal calf serum and antibiotic solution (penicillin-Streptomycin) were purchased from WelGene (Daegu, Korea). Antibodies to p53, p16, caspase3, PARP1 / 2 were obtained from Santa Cruz Biotech, Inc. (Santa Cruz, CA, USA) and p21 antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The GAPDH antibody was obtained from Dr. Kwon Sun - sun of Korea Biotechnology Research Institute. Adriamycin was manufactured by Ildong Pharmaceutical Co., Ltd.

Herbal medicine extract

Herbal medicine extracts were purchased from Plant Extract Bank, Korea Research Institute of Bioscience and Biotechnology. A total of 399 species of herbal extracts (CA01-001 to CA01-100, CA02-001 to CA02-100, CA03-001 to CA03-100, CA04-001 to CA04-100) and 400 water extracts (CW01- 001 to CW01-100, CW02-001 to CW02-100, CW03-001 to CW03-100, and CW04-001 to CW04-100). Each extract was dissolved in dimethylsulfoxide (DMSO) and treated with cells.

3. Cell culture

Human fibroblasts, cervical cancer cells, chondrosarcoma cells, fibrosarcoma cells, and colon cancer cells were used. Each cell was divided into 2 × 10 ⁵ cells in a 100 mm diameter culture dish using DMEM culture medium containing 10% fetal bovine serum and 1% antibiotic (penicillin 10,000 unit / ml, stretomycin 10 mg / ml) And cultured in a 5% carbon dioxide incubator at 37 ° C. When 80-90% of the cells were grown on the bottom of the culture dish, the cells were treated with trypsin-EDTA solution (2.5%), and subcultured. Human fibroblasts were counted every time the cells were transfected and the number of cleavage of the cells was examined. The population doubling (PD) was calculated as PD = log ₂ F / log ₂ I (F = final cell number, I = initial cell number). The cells used in the experiment were those in which the number of cleavage was fewer than 35 in the case of human fibroblasts.

4. Induction of cell aging by treatment with adriamycin

Cell culture was removed and washed twice with DMEM containing antibiotics. Cells were treated with 500 nM adriamycin for 4 hours and then washed twice with DMEM containing antibiotics. After 4 days, senescence-associated β-galactosidase (SA-β-gal) staining was induced to induce cell senescence.

5. Aging-associated beta-galactosidase (SA-beta-gal) active staining

The effects of herbal extracts on cell senescence were examined by SA-β-gal active staining. 12. After 10% of the well plate fetal bovine serum and 1% antibiotic human fibroblasts in DMEM culture medium 500ul containing a is 5x10 ³ dogs, cervical cancer cells, chondrosarcoma cells, fibrosarcoma cells, colon cancer cells such that the dog 1x10 ^4, Divided into each well. After culturing in a 5% CO 2 incubator at 37 ° C for one day, 500 μl of DMEM culture containing 10% fetal bovine serum and 1% antibiotic was added. CA01-018 (Euonymi lignum suberalatu) was treated with 50, 100 ug / ml for 4 days at 37 ℃ in 5% CO2 incubator. After removing the culture medium, the cells were washed twice with 1 × PBS, fixed with 3.7% paraformaldehyde, and then fixed. The cells were stained with SA-β-gal staining solution (40 mM citric acid / phosphate [pH 5.85], 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl ₂ , X-gal 1 mg / 500ul was added. Wrapped in silver foil and reacted at 37 ° C for 16-18 hours. Washed twice with phosphate buffered saline (PBS), and stained with 1% ezine solution for 2 minutes. After washing twice with phosphate buffer, blue-stained cells were observed under an optical microscope. The degree of SA-β-gal activity was expressed as percentage (%) by measuring the number of cells stained blue in the cytoplasm among a total of 100 or more cells.

6. Western blot analysis

The protein (30 μg) was separated by electrophoresis on a 10% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and reacted in TTBS (0.5% Tween 20 in Tris-buffered saline) containing 5% whole milk powder for 1 hour. Nitrocellulose membranes were reacted overnight in a 5% whole milk powder TTBS solution containing primary antibodies against PARP 1/2, caspase 3, p53, p21 or p16. After washing three times with TTBS solution, they were reacted with secondary antibody conjugated with horseradish peroxidase for 3 hours. The membranes were washed 5 times for 5 min with TTBS and protein expression was measured using enhanced chemiluminescence solution. The amount of specific protein reacted with each antibody was measured using a LAS-3000 imaging device (Fujifilm Corp., Stanford, CT, USA). The same amount of protein used in each experiment was confirmed using glyceraldehyde-3-phosphate dehydrogease (GAPDH) antibody.

7. Cell Count

A 12 well plate 10% fetal bovine serum and 1% antibiotic DMEM culture solution 500 ul containing the the fibroblasts to the 5000 / well, cervical cancer cells, chondrosarcoma cells, fibrosarcoma cells and colon cancer cells more 1 × 10 ⁴ And then dispensed into each well. After culturing in a 5% CO 2 incubator at 37 ° C for one day, 500 μl of a DMEM culture containing 10% fetal bovine serum and 1% antibiotic was added. CA01-018 (Euonymi lignum suberalatu) was treated with 50, 100 ug / ml for 4 days at 37 ℃ in 5% CO2 incubator. After the culture medium was removed, the cells were removed from the bottom with 2.5 x trypsin-EDTA solution, and the number of cells was measured using a hemocytometer.

8. Cell protein extraction

Each cell was divided into 2 × 10 ⁵ cells in a 60 mm culture dish and cultured at 37 ° C. in a 5% carbon dioxide incubator. Cell culture medium was changed and herbal extract fraction was treated for 4 days by concentration. After the culture solution was removed, it was washed once with phosphate buffer solution. 50 μl of a cell lysis solution (25 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% Tryton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium vanadate, 5 mM NaF, protease inhibitor or 1 mM PMSF) . The solution and cells were collected using a cell scrape and then transferred to a micro-incision tube. The solution was shaken every 10 minutes while reacting on ice for 30 minutes. The supernatant was transferred to a new tube by spinning at 12,000 xg for 10 minutes. The amount of protein in the solution was determined by bicinchoninic acid (BCA) method (Pierce Biotechnology Inc., Rockford, IL, USA) using bovine serum albumin as a standard protein.

9. Statistical processing

Each experiment was repeated three times at three times at a time to obtain mean and standard deviation. Statistical significance was analyzed by Student's t-test, and p value <0.05 (p <0.05) was considered significant.

10. Results

Among 799 herbal medicine extracts in human fibroblasts, SA-β-gal activity staining was performed in order to extract an extract effective for promoting cell senescence by adriamycin. The efficacy of the substances was compared by treating dimethylsulfoxide (DMSO), a solvent used to dissolve each material, and N-acetylcysteine (NAC), a substance known to inhibit cell senescence. Each experiment was performed three times in 3 replicates. Among them, CA01-018 (Euonymi lignum suberalatu, ear-wolf) was identified as an herbal extract which increases SA-β-gal activity. To further confirm the SA-β-gal activity increasing effect of CA01-018, fibroblasts were treated with adriamycin and each herbal extract was treated with 50 and 100 μg / ml to perform SA-β-gal activity staining. As a result, SA-β-gal active staining was increased in a concentration-dependent manner (FIGS. 1A and 1B). Therefore, it was confirmed that CA01-018 promotes cell aging of fibroblasts by treatment with anticancer agents. Western blot analysis was performed to investigate whether CA01-018 promotes cell senescence by affecting the expression of PARP1 / 2, caspase 3, or cancer suppressor genes p53 and p16. As a result, no change in the expression of PARP1 / 2, caspase 3, p53 and p16 protein was observed, suggesting that promotion of cell senescence by the extract does not occur through apoptosis or expression of a cancer-suppressing gene (Fig. 1C).

< Example  2> in human fibroblasts CA01 -018 cytotoxicity studies

1. Analysis of 3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide (MTT) from 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide

Human fibroblasts were dispensed into each well of a 96-well plate in a volume of 100 μl in 500-well culture media. One day later, CA01-018 (Euonymi lignum suberalatu) was treated with concentration (10, 25, 50, 100 ul / ml). And cultured in a 5% CO 2 incubator at 37 ° C for 4 days. 50 μl of 0.1% MTT solution was added to each well and reacted in a 5% CO 2 incubator at 37 ° C for 3 hours. After removing the culture solution and MTT solution, 100 μl of DMSO was added to dissolve the crystals. Absorbance was measured at 550 nm using a microplate reader.

2. Results

Before investigating the cell senescence promoting effect of CA01-018 in cancer cells, each extract was examined for cytotoxicity in human fibroblasts. When human fibroblasts were treated with increasing concentrations of CA 01-018 to 100 ug / ml, there was no significant cytotoxicity compared to DMSO (FIG. 2A). In human fibroblasts, cell senescence was promoted or SA-β-gal active staining was performed. As a result, it was confirmed that SA-β-gal activity staining was increased in a concentration-dependent manner, thereby promoting cell senescence (FIGS. 2B and 2C). The expression of PARP1 / 2, caspase3, p53 and p16 protein was examined after 20 hours of treatment with CA01-018 (Fig. 2D).

< Example  3> human cervical cancer cells ( HeLa ), Chondrosarcoma ( HTB94 ), Fibrosarcoma  cell( HT1080 ), Colon cancer cells ( HCT116 WT , HCT116 p53 - / -, HCT116 p21 -/-)in p53 , p21, p16 Investigation of the expression of

The expression of p53, p21, p16 tumor suppressor gene in each cancer cell was investigated by Western blotting before CA01-018 cancer cell activation promoting activity was examined. The expression of p53 and p21 in HTB94 cells, the expression of p53 in HT1080 cells, the expression of p53 and p21 in HCT116 WT cells, and the expression of p53 and p21 in HCT116 p53 P21 expression was observed in HCT116 p21 - / - cells, and p53 expression was observed in HCT116 p21 - / - cells (Fig. 3).

< Example  4> in cancer cells CA01 -018 cell aging promotion effect investigation

1. Enhancement of cellular senescence of extracts from human cervical cancer cells (HeLa)

The cytotoxic effect of CA01-018 (Euonymi lignum suberalatu) on human cervical cancer cells was investigated by SA-β-gal staining. Adriamycin, which is known to inhibit growth of cancer cells and accelerate cell aging of cancer cells, was treated with a positive control group of dimethylsulfoxide (DMSO) as a negative control to dissolve herbal extracts, and the effect of each herbal medicine extract was compared. As a result, it was confirmed that SA-β-gal active staining was increased in a concentration-dependent manner to promote cell senescence (FIGS. 4A and 4B). Since cell growth is reduced when cell senescence is promoted, cell growth by each extract is counted. As a result, cell growth also decreased in a concentration-dependent manner of CA 01-018 (FIG. 4C). The cytotoxic and cell growth inhibitory effect of CA01-018 was higher than that of adriamycin, which is well known as an anticancer drug. After each extract treatment, the degree of expression of PARP1 / 2, caspase3, and p53 protein was examined by western blotting, but there was no difference (Fig. 4D).

2. Promoting cell aging of extracts from human chondrosarcoma cells (HTB94)

The effect of CA01-018 (Euonymi lignum suberalatu) on cell senescence was examined by SA-β-gal staining. CA01-018 increased SA-β-gal activity staining in a concentration-dependent manner, and confirmed that it promoted cell senescence (FIGS. 5A and 5B). Similarly, cell growth was also decreased in a CA 01-018 concentration-dependent manner. After CA01-018 treatment, expression of PARP1 / 2, caspase3, and p53 protein was examined by western blot method and no difference in expression of each protein was observed (Fig. 5D).

3. Promoting cell senescence of extracts from human fibrosarcoma cells (HT1080)

The cell senescence-promoting effect of CA01-018 (Euonymi lignum suberalatu) was examined by SA-β-gal staining. CA01-018 increased SA-β-gal activity staining in a concentration-dependent manner, and confirmed that cell senescence was promoted (FIGS. 6A and 6B). Similarly, cell growth also decreased in a concentration-dependent manner of CA 01-018 (FIG. 6C). After treatment with CA01-018, the degree of PARP1 / 2, caspase3, and p53 protein expression was not significantly changed (Fig. 6D).

4. Promoting cell senescence of extracts from human colorectal cancer cells (HCT116 WT, HCT116 p53 - / -, HCT116 p21 - / -)

Human colonic cancer cells were examined for their ability to stimulate cellular senescence of CA01-018 (Euonymi lignum suberalatu, ear) in wild type HCT116 cells and HCT116 mutants lacking the cancer suppressor genes p53 and p21, respectively.

In HCT116 WT cells, CA01-018 increased SA-β-gal activity staining in a concentration-dependent manner, confirming promoting cell senescence (FIGS. 7A and 7B). Cell growth was also decreased in a CA 01-018 concentration-dependent manner (Fig. 7C). The expression levels of PARP1 / 2, caspase3, and p53 protein were examined and the expression of each protein was not observed (FIG. 7D).

Similarly, in HCT116 p53 - / - cells, CA01-018 increased SA-β-gal activity staining in a concentration-dependent manner and promoted cell senescence (FIGS. 8A and 8B). Cell growth also decreased in a CA 01-018 concentration-dependent manner (Fig. 8C). The degree of expression of PARP1 / 2, caspase3, and p16 protein by CA01-018 treatment was not significantly changed (Fig. 8D).

In HCT116 p21 - / - cells, CA01-018 (Euonymi lignum suberalatu, ear bulb) did not increase SA-β-gal activity staining (FIGS. 9A and 9B). Cell growth was decreased in a CA 01-018 concentration-dependent manner (Fig. 9C). The expression of PARP1 / 2 and caspase3 was decreased by CA01-018 (Euonymi lignum suberalatu, ear bulb) (Fig. 9D).

Claims (6)

A pharmaceutical composition for the prevention or treatment of colorectal cancer, which comprises an extract of ethanol extract of ear skin as an active ingredient, and the ethanol extract of the skin extract promotes senescence of colon cancer cells to inhibit the growth of colon cancer cells. delete delete The method according to claim 1, wherein the senescence of the colorectal cancer cells is promoted by measuring an increase in senescence-associated beta-galactosidase (SA-beta-gal) activity. A pharmaceutical composition for therapeutic use.

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