KR20210020849A - Pharmaceutical composition for preventing or treating ovarian cancer or breast cancer comprising chrysophanol - Google Patents

Abstract

The present invention relates to a composition including chrysophanol as an active ingredient, and particularly, to a pharmaceutical composition for preventing or treating ovarian cancer or breast cancer including chrysophanol as an active ingredient, or a health-aid food composition for preventing of alleviating ovarian cancer or breast cancer including chrysophanol as an active ingredient. The composition including chrysophanol as an active ingredient according to the present invention is a substance extracted from plants to minimize in vivo toxicity or side-effects as compared to a chemically synthesized product, inhibits proliferation of ovarian cancer and breast cancer cells, increases the number of dead cells in ovarian cancer and breast cancer cell lines in a dose-dependent manner, and provides a synergic effect of apoptosis when being used in combination with a target inhibitor inhibiting PI3K/AKT and MAPK signaling paths, and thus can be used widely in various industrial fields including life science, medicine and pharmacy.

Description

Pharmaceutical composition for preventing or treating ovarian cancer or breast cancer comprising chrysophanol {Pharmaceutical composition for preventing or treating ovarian cancer or breast cancer comprising chrysophanol}

The present invention relates to a composition comprising chrysophanol as an active ingredient, and more specifically, a pharmaceutical composition for the prevention or treatment of ovarian cancer or breast cancer comprising chrysophanol as an active ingredient, and chrysophanol as an active ingredient. It relates to a health functional food composition for preventing or improving ovarian cancer or breast cancer comprising.

Ovarian cancer has a very high mortality rate versus incidence due to the lack of early diagnosis technology and resistance to chemotherapy (Kobayashi et al. , Journal of Translational Medicine, 2014). Currently, after tumor reduction treatment, platinum-based, taxane-based} chemotherapy or radiation therapy is considered the standard of ovarian cancer treatment, but the recurrence rate is approximately 70% in terminal ovarian cancer patients (Capriglione et al. , Medical Oncology, 2017). ).

Breast cancer has the 2nd incidence rate among malignant tumors in women worldwide, and is the 2nd most fatal disease with a mortality rate of 1 in 8 people throughout life (Siegel et al., CA Cancer J., 2018, 68: p. .7-30). Although mutations in the BRCA gene are known to be the leading cause of breast cancer, the number is only 5-10% of breast cancer patients. In addition, it is known that women who do not lactate or who have a short lactation period have a high incidence of breast cancer. (Collaborative Group on Hormonal Factors in Breast, C. Lancet, 2002, 360 : p.187-195.) In general, for breast cancer, a treatment that surgically removes the entire tumor is used. However, until recently, improved surgical methods or chemotherapy have a problem that they do not show any effect in patients with advanced stage and only worsen side effects (Aghapour et al. , Biochem. Biophy. Res. Commun, 2018, 500: p.860: p.860) -865).

On the other hand, chrysophanol is an anthraquinone-based natural product mainly contained in rhubarb used as an ingredient in herbal medicine. While representative anthraquinone-based compounds such as emidine and lane are known to induce apoptosis and inhibit invasiveness of various carcinomas, the anticancer effect of chrysophanol, in particular, the treatment effect of ovarian cancer and breast cancer, which are representative gynecological diseases, is still Has not been reported.

Under the above-described technical background, the present inventors made diligent efforts to develop new ovarian cancer and breast cancer using natural substances, and as a result, chrysophanol inhibited cell proliferation of ovarian cancer or breast cancer and confirmed that it has an effect of inducing cell death. The invention was completed.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ovarian cancer or breast cancer comprising chrysophanol as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or improving ovarian cancer or breast cancer comprising chrysophanol as an active ingredient.

Another object of the present invention is to provide a method of inhibiting the proliferation ability of ovarian cancer or breast cancer cell lines by using the pharmaceutical composition or health functional food composition.

Another object of the present invention is to provide a method of improving the killing effect of ovarian cancer or breast cancer cell lines by using the pharmaceutical composition or health functional food composition.

The present invention provides a pharmaceutical composition for preventing or treating ovarian cancer or breast cancer comprising chrysophanol as an active ingredient.

The present invention also provides a health functional food composition for preventing or improving ovarian cancer or breast cancer comprising chrysophanol as an active ingredient.

The present invention also provides a method of inhibiting the proliferation ability of ovarian cancer or breast cancer cell lines by using the pharmaceutical composition or health functional food composition.

The present invention also provides a method of improving the killing effect of ovarian cancer or breast cancer cell lines by using the pharmaceutical composition or health functional food composition.

The composition comprising chrysophanol according to the present invention as an active ingredient is extracted from plants and can minimize toxicity or side effects in the body compared to chemical compounds, inhibit proliferation of ovarian cancer and breast cancer cells, and dose-dependently, ovarian cancer and In addition to increasing the number of dead cells in breast cancer cell lines, synergistic effects of cell death can be obtained when combined treatment with target inhibitors that inhibit PI3K/AKT and MAPK signaling pathways, and various industries such as life science and medicine It can be widely used in

Figure 1 shows the results of measuring the effect of reducing the proliferation of ovarian cancer and breast cancer cells by chrysophanol.
Figure 2 shows the results of measuring the effect of inducing ovarian cancer and breast cancer cell death by chrysophanol.
3 shows the results of measuring changes in oxidative stress in ovarian cancer and breast cancer cells by chrysophanol.
Figure 4 shows the results of measuring the mitochondrial dysfunction induction effect in ovarian cancer and breast cancer cells by chrysophanol.
5 shows the results of analyzing the regulation of signaling mechanisms in ovarian cancer and breast cancer by chrysophanol.
6 shows the results of measuring changes in ovarian cancer and breast cancer cell proliferation capacity according to treatment with chrysophanol and a signaling pathway inhibitor.
7 shows the results of measuring the effect of reducing the ovarian cancer cell migration ability by chrysophanol.
Figure 8 shows the anticancer mechanism of chrysophanol against ovarian cancer.
9 shows the mechanism of anticancer action of chrysophanol on breast cancer cells.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In the present invention, chrysophanol inhibited the proliferation of ovarian cancer or breast cancer cell lines and revealed a mechanism of cell death (FIGS. 8 and 9 ).

The present invention relates to a pharmaceutical composition for the prevention or treatment of ovarian cancer and breast cancer comprising chrysophanol as an active ingredient in one aspect.

The present invention relates to a health functional food composition for preventing or improving ovarian cancer and breast cancer comprising chrysophanol as an active ingredient from another viewpoint.

The term "composition" as used in the present invention is considered to include not only products containing specific ingredients, but also any products made directly or indirectly by the combination of specific ingredients.

In the present invention, the ovarian cancer or breast cancer cells may be cells derived from mammals, and the mammals are rodents (eg, mice, rats, hamsters, gerbils, and guinea pigs), right heads (eg For example, cattle, sheep, pigs, goats, deer, giraffes and antelopes), base titles (e.g. horses, donkeys, rhinos and tapirs), carnivores (e.g. dogs, cats, tigers, wolves, foxes, lions) , Cheetahs, leopards, raccoons, badgers, cougars, jaguars and wildcats), hare (rabbits and crying rabbits), carnivores (e.g. hedgehogs, moles and solenoidons) and primates (e.g. chimpanzees, orangutans , Gorilla, bono bono, Japanese monkey, rhesus monkey).

In the present invention, the pharmaceutical composition may be characterized in that it contains a pharmaceutically acceptable carrier, wherein the carrier is an ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer material, water, salt, Electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol and wool paper, characterized in that at least one selected from the group consisting of I can.

In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration. It can be characterized in that it further contains any one or more auxiliary agents selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity modifiers, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , It may be characterized in that it has a formulation selected from the group consisting of gels and powders.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the severity of symptoms, weight, age, sex, mode of administration and time of administration of the patient, and generally skilled physicians are effective in the desired treatment or prevention. The dosage can be easily determined.

In the present invention, the health functional food refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No. 6722, and contains nutrients for the structure and function of the human body. It refers to foods consumed for the purpose of controlling or obtaining beneficial effects for health purposes such as physiological effects.

The health functional food composition of the present invention may be formulated into a formulation of a general health functional food known in the art, and granules, tablets, pills, suspensions, emulsions, syrups, gums, teas, jellies, various beverages, and drinks. , Alcoholic beverages, etc., and there is no particular limitation on the kind of the health functional food.

The health functional food composition of the present invention is in any herbal form suitable for administration to an animal body, including the human body, more specifically any form conventional for oral administration, for example, food or feed, additives and adjuvants for food or feed, It may be in a solid form such as fortified food or feed, tablets, pills, granules, capsules and foam formulations, or in liquid form such as solutions, suspensions, emulsions, beverages, pastes, etc., and nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, It may contain a preservative or the like, and these ingredients may be used independently or in combination.

According to the present invention, the chrysophanol may be characterized in that it regulates PI3K/AKT and MAPK signaling mechanisms.

In addition, according to the present invention, it may be characterized in that it further comprises a target inhibitor that inhibits the PI3K/AKT and MAPK signaling pathways.

According to the present invention, the chrysophanol may prevent or treat ovarian cancer and breast cancer through induction of oxidative stress.

According to the present invention, the chrysophanol may prevent or treat ovarian cancer and breast cancer through induction of mitochondrial dysfunction.

The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent.

In the present invention, cisplatin or paclitaxel may be further included, but the present invention is not limited thereto, and a chemotherapeutic agent used as a therapeutic agent for ovarian cancer or breast cancer may be further included.

In another aspect, the present invention relates to a method of inhibiting the proliferation ability of a mammalian-derived ovarian cancer cell line excluding humans or a mammalian-derived breast cancer cell line excluding humans by using a composition comprising chrysophanol as an active ingredient.

In another aspect, the present invention relates to a method for improving the killing effect of a mammalian-derived ovarian cancer cell line excluding humans or a mammalian-derived breast cancer cell line excluding humans by using a composition comprising chrysophanol as an active ingredient.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Experiment method

Experimental animals and cell culture

ES2 and OVCAR3 cells were purchased from the American Type Culture Collection and used.For monolayer culture of cells, ES2 was used in McCoy's 5A medium, and OVCAR3 was used in RPMI-1640 medium containing 25 mM HEPES, and 10% fetal bovine serum. , FBS) were mixed together and used. Breast cancer cell lines BT-474 and MCF-7 were purchased from the Korea Cell Line Bank (KCLB), and normal mammary cells, MCF-12A, were purchased from the American Type Culture Collection and used. For monolayer culture of breast cancer cells, 10% fetal bovine serum (FBS) was added to RPMI1640 (+25mM HEPES) medium, and normal mammary cells were 20 ng/ml in DMEM/F12 medium. HEGF, 0.01mg/ml bovine insulin, 500ng/ml hydrocortisone, and 5% horse serum were mixed in culture medium.

Experiment material

Chrysophanol, a candidate composition, was purchased and used from Sigma-Aldrich, Inc, and phospho-AKT (Ser ⁴⁷³ ), ERK1/2 (Thr ²⁰² /Tyr ²⁰⁴ ), JNK (Thr ¹⁸³ /Tyr ¹⁸⁵ ), P70S6K (Thr ⁴²¹ /Ser ⁴²⁴ ), P90RSK (Thr ⁵⁷³ ), S6 (Ser ^{235 /236} ), P38 (Thr ¹⁸⁰ /Tyr ¹⁸² ) protein and total-AKT, ERK1/2, JNK , P70S6K, P90RSK, S6, P38 antibodies were purchased from Cell Signaling Techonology. In addition, in order to investigate the effect of inhibiting the target signaling process, PI3K inhibitor LY294002 was purchased from Cell Signaling Technology, ERK1/2 inhibitor U0126, P38 inhibitor SB203580 and JNK inhibitor SP600125 were purchased from Enzo Life Science. I did.

BrdU Cell proliferation ability analysis

To confirm the effect of chrysophanol on the proliferation ability of ovarian cancer cells and breast cancer cells, 5×10 ³ cells cultured under FBS starvation and 100 μl of medium were dispensed into 96 wells and treated with chrysophanol dose-dependently. After incubation for 48 hours, the experiment was performed according to the manufacturer's manual using the BrdU kit (Cat No: 1167229001, Roche). After incubation for 48 hours, 10 μM BrdU was additionally added to each well and incubated for 2 hours in _{a 37° C./5% CO 2 incubator.} The cells are labeled with BrdU and the cells are fixed, incubated with anti-BrdU-POD solution for 90 minutes at room temperature, washed three times, and finally reacted with 100 μl of 3,3',5,5'-tetramethylbenzidine substrate. And, using an ELISA reader, the absorbance within 370 nm and 492 nm was measured to analyze the cell proliferation ability.

Immunofluorescence Cell proliferation used Marker Confirm

3×10 ⁴ BT-474 and MCF-7 cells were dispensed and cultured in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea), and then further cultured in FBS starvation state for 24 hours. After treatment with 100 μM for 24 hours, the cells were fixed with methanol for 10 minutes, and 2 μg/ml of PCNA antibody was treated and incubated at 4° C. for 16 hours. After that, after washing twice with PBS containing 0.1% BSA (bovine serum albumin), goat anti-mouse IgG Alexa 488 (catalog number: A-11001, Invitrogen, Carlsbad, CA, USA) was used as the secondary antibody. It was diluted 1:200 in antibody dilution buffer and incubated for 1 hour at room temperature. After washing the cells with 0.1% BSA-PBS, DAPI staining was additionally performed so that the nuclei could be observed simultaneously. After the end of the experiment, the cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.

Annexin V and propidium Apoptosis analysis through iodide staining

In order to confirm the killing effect of ovarian cancer and breast cancer cells by chrysophanol, an experiment was conducted using FITC Annexin V Apoptosis Diagnostic Kit I (BD Biosciences). First, 5×10 ⁵ cells were cultured in 6 wells and further cultured in FBS starvation for 24 hours when the cells were filled in a 70-80% culture dish. Thereafter, chrysophanol was dose-dependently treated and cultured in a 37° C./5% CO 2 incubator for 48 hours. Thereafter, the cells were removed from the culture dish using trypsin, washed with PBS, and the cells were slowly mixed using 1 mL of 1× binding buffer and centrifuged to obtain a cell pellet, followed by 200 μL of 1× binding buffer. After cell suspension culture, 100 μL was added to a brown 1.5 mL tube, and 5 μL of Annexin V and 5 μL of PI were mixed together, and the cells were left at room temperature for 15 minutes to stain. Thereafter, 400 μL of 1× binding buffer was added, and the stained solution was transferred to a 5 mL FACS tube, and fluorescence intensity was analyzed using a flow cytometer to measure the number of dead cells.

TUNEL DNA through assay Segmentation Measure

After culturing 3×10 ⁴ BT-474 and MCF-7 cells in a confocal dish (catalog number: 100350, SPL), they were further cultured in serum starvation for 24 hours and treated with 100 μM of chrysophanol for 48 hours. . Thereafter, the cells were air-dried and incubated for 1 hour at room temperature with 4% paraformaldehyde to fix the cells. The fixed cells were rinsed once with PBS and incubated on ice for 2 minutes using a solution containing 0.1% sodium citrate and 0.1% Triton X-100. Next, using the TUNEL staining mixture included in In Situ Cell Death Detection Kit, TMR red (Roche), it was incubated for 1 hour in a 37°C/5% CO2 incubator. Finally, after rinsing with PBS and staining with DAPI, cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.

DCFH -Analysis of active oxygen formation using DA

Reactive oxygen formation analysis was performed through 2',7'-dichlorofluorescin diacetate (DCFH-DA, Sigma-Aldrich). When the 100π culture dish was filled with 70 to 80% cells, the cells were removed from the culture dish using trypsin, washed with PBS, and centrifuged to obtain a cell pellet. Cells were treated with 10 μM DCFH-DA and incubated for 30 minutes in a 37° C./5% CO 2 incubator. Then, after washing with PBS, chrysophanol was dose-dependently treated and cultured in a _{37°C/5% CO 2 incubator for 1 hour.} The cells were centrifuged again, washed with PBS, and then analyzed for fluorescence intensity using a flow cytometer.

Lipid peroxidation analysis

Click-iT lipid peroxidation imaging kit (Invitrogen) was used to analyze the degree of lipid peroxidation in ovarian cancer and breast cancer cells. 3 × 10 ⁴ cells were cultured in a confocal dish with 300 μl of a medium containing 10% FBS, and then incubated with 100 μM of chrysophanol in an incubator at 37° C. for 2 hours. Cells were fixed with 3.7% formaldehyde, premeablized with 0.5% Triton X-100, and fluorescently stained with Alexa Fluor 488 Azide for 30 minutes at room temperature. Finally, after rinsing with PBS and staining with DAPI, cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.

Fluo -4 and Rhod With -2 Intracellular Ca ^{2 +} Concentration measurement

In order to confirm the effect of chrysophanol on the intracellular Ca ^{2 + concentration, 5 × 10 5} cells were first cultured in 6 wells and further cultured in FBS starvation for 24 hours when the cells were filled in a 70-80% culture dish. Thereafter, chrysophanol was treated and cultured in a 37° C./5% CO 2 incubator for 48 hours. Thereafter, the cells were removed from the culture dish using trypsin, and Fluo-4 AM (Invitrogen) was incubated for 20 minutes in an incubator at 37°C. The stained cells were washed once with PBS and analyzed for fluorescence intensity using a flow cytometer. To measure the mitochondrial-specific Ca ^{2 +} concentration, Rhod-2 AM (Invitrogen) was diluted in HBSS and incubated at 4° C. for 30 minutes. The stained cells were washed with HBSS and analyzed for fluorescence intensity using a flow cytometer.

JC Mitochondria through -1 staining Membrane potential Measure

JC-1 mitochondrial membrane potential (MMP) change was measured using a mitochondria staining kit (Cat No: CS0390, Sigma-Aldrich). 5×10 ⁵ ovarian cancer and breast cancer cells were cultured in 6 wells and further cultured in FBS starvation for 24 hours when the cells were filled in 70-80% culture dishes. Thereafter, chrysophanol was dose-dependently treated (0, 20, 50, 100 μM) and cultured in a _{37° C./5% CO 2 incubator for 48 hours.} Thereafter, the cells were removed from the culture dish using trypsin and centrifuged to obtain a cell pellet. The cells were released in JC-1 staining solution and incubated for 20 minutes in a 37°C/5% CO2 incubator. The stained cells were centrifuged again, washed with 1x JC-1 staining buffer, and the fluorescence intensity was analyzed using a flow cytometer.

Protein expression analysis ( Western Blot )

Ovarian cancer and breast cancer cells were treated with chrysophanol, and then total protein was extracted, and the protein was quantified by Bradford protein assay (Bio-Rad, Hercules, CA, USA). After that, the extracted protein was denatured at 95°C for 5 minutes, electrophoresis was performed using 10% SDS/PAGE gel, transferred to a nitrocellulose membrane, and the primary antibody and the secondary antibody were incubated sequentially, followed by chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) reagent, and ChemiDoc EQ system and Quantity One software (Bio-Rad) instrument were used to analyze the expression of the target protein.

cell Invasive analysis

To confirm the effect of chrysophanol on the cellular invasiveness of ovarian cancer cells, 1×10 ⁵ ovarian cancer cells cultured under FBS starvation conditions after coating Matrigel on migration culture dishes were inserted into 8-mm transwell inserts (Corning, Inc. ., Corning, NY, USA), treated with chrysophanol, incubated for 12 hours (ES2) or 16 hours (OVCAR3), and then fixed the cells transferred trans-membrane with methanol for 10 minutes, dried and Hematoxylin (Sigma Aldrich, Inc.) staining was performed for 30 minutes and washed. The membrane was placed on a slide glass and fixed with a Permount solution, and the cell migration ability was observed, photographed and calculated with an optical microscope DM3000 (Leica). In addition, in order to analyze the migration change of cells through Ibidi migration inserts, ^{70 μl of a medium containing 2×10 5} cells/ml of ovarian cancer cells was cultured in inserts, and starvation was maintained for 24 hours. After removing the inserts and treating with chrysophanol, the average length between cell groups was measured.

Statistical analysis

The mean and standard error were calculated using SAS (statistical analysis system) statistical program, and one-way ANOVA was performed. A significance test was conducted at the P <0.05 level.

Results and Discussion

To chrysophanol Inhibition of proliferation and death of ovarian cancer and breast cancer cells

To determine whether chrysophanol affects the proliferation of ovarian cancer cell lines, ES2 cells and OVCAR3 cells, dose-dependent treatment with chrysophanol (0, 5, 10, 20, 50, 100 μM) and then BrdU ELISA assay I did. As a result, the experimental group treated with chrysophanol showed a tendency to decrease cell proliferation in a dose-dependent manner compared to the control group treated with DMSO (FIG. 1A). 100 μM of chrysophanol decreased the proliferation capacity of ES2 cells and OVCAR3 cells by 52% ( p <0.001) and 55% ( p <0.001), respectively. In addition, in order to determine the effect of chrysophanol on the growth of breast cancer cell lines BT-474 and MCF-7 cells, it was cultured for 48 hours in a culture medium treated with chrysophanol dose-dependently (0, 5, 10, 20, 50, 100 μM). Then, the degree of cell proliferation was measured using BrdU. As a result, it was confirmed that the cell proliferation significantly decreased from 20 μM in both cell lines (Fig. 1B). On the other hand, it was confirmed that the normal mammary cell line MCF-12A did not significantly decrease the degree of cell proliferation even when treated with the same concentration of chrysophanol (FIG. 1C). According to these results, the expression rate of PCNA, an intranuclear proliferation marker, was confirmed in breast cancer cell lines by treatment with 100 μM concentration of chrysophanol, and it was confirmed that the expression decreased in the cell line treated with chrysophanol compared to the cell line treated with only solvent. (Fig. 1D).

Next, to determine whether chrysophanol can induce the death of ovarian cancer cells, FITC-conjugated Annexin V and PI staining were performed on ES2 cells and OVCAR3 cells. As a result, chrysophanol induced the death of ES2 and OVCAR3 cells in a dose-dependent manner (FIG. 2A). When ES2 cells were treated with 100 μM of chrysophanol, the proportion of cells stained with both Annexin V and PI increased about 8 times ( p <0.001) compared to the control group. Similarly, 100 μM of chrysophanol increased the death of OVACR3 cells by about 11 times ( p <0.001). These results suggest that chrysophanol may inhibit the proliferation of ovarian cancer cells and have anticancer effects. In addition, in order to confirm the effect of chrysophanol on the apoptosis of breast cancer cell lines BT-474 and MCF-7, a culture medium treated with chrysophanol dose-dependently (0, 10, 20, 50, 100 μM) was treated for 48 hours. Afterwards, the number of dead cells was confirmed through Annexin V & PI staining. As a result, cell death was gradually increased by chrysophanol in both cell lines (FIG. 2B ). On the other hand, in MCF-12A cells, which is a normal mammary cell line, significant apoptosis was shown only when the highest concentration of chrysophanol (100 μM) was treated. Decreased (Fig. 2C). It was confirmed through the TUNEL assay that chrysophanol occurs by inducing segmentation in the DNA of the breast cancer cell line (Fig. 2D).

To chrysophanol Inducing oxidative stress in ovarian cancer cells

To determine whether chrysophanol can induce oxidative stress in ovarian cancer cells, the intensity of DCF green fluorescence was measured (FIG. 3A). ES2 and OVCAR3 cells were cultured for 2 hours in a medium containing chrysophanol (0, 20, 50, 100 μM). In addition, 0.03% hydrogen peroxide was used as a positive control. The production of ROS in ES2 cells was dose-dependently increased by chrysophanol and increased by about 24 times ( p <0.001) at the highest concentration. However, in OVCAR3 cells, the production of ROS by chrysophanol was not significantly increased. Lipid peroxidation, a sub-physiological phenomenon caused by intracellular ROS production, also showed a similar tendency (FIG. 3B). Chrysophanol did not change the degree of lipid peroxidation in OVCAR3 cells, but increased lipid peroxidation in ES2 cells by about 12 times (p <0.001) compared to the control. In addition, free radicals were increased in both BT-474 and MCF-7 by chrysophanol (FIG. 3C), and lipid phosphorylation in the cytoplasm was observed (FIG. 3D).

To chrysophanol Induction of mitochondrial dysfunction in ovarian cancer cells

Dysfunction caused by excessive calcium ion influx into mitochondria is a representative physiological mechanism leading to cell death. Therefore, fluo-4 and rhod-2 staining were performed, respectively, in order to measure changes in the concentration of intraocular and mitochondrial-specific calcium ions by chrysophanol. As a result, it was confirmed that chrysophanol (0, 20, 50, 100 μM) did not significantly affect the calcium ion concentration in ES2 cells and OVCAR3 cells (Fig. 4A). However, the concentration of mitochondrial-specific calcium ions stained with rhod-2 increased dose-dependently with chrysophanol treatment (FIG. 4B). By 100 μM of chrysophanol, mitochondrial-specific calcium ions increased 8.5-fold ( p <0.001) and 2.5-fold ( p <0.001), respectively, in ES2 cells and OVCAR3 cells. Next, JC-1 staining was performed after treatment with chrysophanol dose-dependently (0, 20, 50, 100 μM) in order to confirm the change of mitochondrial membrane potential by chrysophanol. As a result, it was found that the mitochondrial membrane potential in the ovarian cancer cell line was decreased by chrysophanol (Fig. 4C). 100 μM of chrysophanol reduced mitochondrial membrane potential in ES2 and OVCAR3 cells by 4.2 times ( p <0.001) and 2.7 times ( p <0.001), respectively, compared to the control group. These results suggest that chrysophanol may induce dysfunction due to increased influx of calcium ions into mitochondria in ovarian cancer cells. In addition, it was confirmed that mitochondrial membrane potential was reduced by treatment with chrysophanol in breast cancer cell lines through JC-1 staining (FIG. 4D). In addition, it was confirmed that calcium ions in the cytoplasm increased according to the dose-dependent treatment of chrysophanol in both BT-474 and MCF-7 cells (Fig. 4E). It was also confirmed that the action of this chrysophanol on mitochondria increases the expression of mitochondrial-dependent cell death proteins Bax, Bak, and cytochrome c (FIG. 4F).

Chrysophanol and Analysis of changes in proliferation of ovarian cancer cells through signal transduction mechanism inhibitors

To analyze the effect of chrysophanol on the activation of MAPK and PI3K/AKT signaling pathways, which are representative signaling pathways involved in the survival and proliferation of ovarian cancer cells, chrysophanol was time-dependent (0, 5, 15, 30, 60 minutes), the phosphorylation of the signaling pathway protein was confirmed through Western blot (Figs. 5A-5F). As a result, in the case of ERK1/2, the MAPK signaling protein, phosphorylation increased after 5 minutes treatment with chrysophanol, and then decreased with time. In addition, P90RSK, a sub-signaling protein of ERK1/2, showed the highest degree of phosphorylation at a slower 15 minutes (ES2) or 60 minutes (OVCAR3). The phosphorylation of another MAPK protein, JNK and its sub-signaling protein, c-Jun, in ovarian cancer cells was also highest after 5 or 60 minutes of chrysophanol treatment. In addition, phosphorylation of AKT was highest when treated with chrysophanol for 5 minutes and decreased thereafter.P70S6K, a lower signaling protein of AKT, also showed the highest degree of phosphorylation when treated with chrysophanol for 5 minutes in ES2 and 60 minutes in OVCAR3. Gradually increased to. In addition, in breast cancer cell lines BT-474 and MCF-7, MAPK and PI3K/AKT pathway proteins were detected through Western blot in order to confirm the change in the activity of intracellular signaling mechanisms by chrysophanol treatment (Fig. 5G- 5L). As a result, phosphorylation of ERK1/2 was decreased by chrysophanol in both cell lines, phosphorylation of P38 and JNK decreased in BT-474, whereas increased in MCF-7, and dose-dependent treatment of chrysophanol was AKT, P70S6K. , It has been shown to reduce all of the phosphorylation of S6. Through these results, it was confirmed that chrysophanol can regulate phosphorylation of cell signaling proteins in breast cancer cell lines.

Next, to investigate whether the combination treatment with an inhibitor of the activity of PI3K/AKT and MAPK pathway proteins and chrysophanol causes changes in ovarian cancer cell proliferation, chrysophanol (100 μM) and LY294002 (AKT inhibitor, 20 μM) , U0126 (ERK1/2 inhibitor, 20 μM), SP600125 (JNK inhibitor, 20 μM), SB203580 (P38 inhibitor, 20 μM) were co-treated (Fig. 6A). As a result, in ES2 cells, the antiproliferative effect of chrysophanol was further enhanced by U0126 treatment ( p <0.05) and alleviated by SB203580 treatment ( p <0.05). In OVCAR3 cells, the proliferative capacity decreased by chrysophanol was further decreased in combination treatment with all inhibitors. In addition, in order to analyze the signal transduction mechanism regulated by the treatment of chrysophanol in breast cancer cell lines, the results of confirming the change in the proliferative capacity of the cell lines were confirmed by treatment with chrysophanol and LY294002, U0126, SB203580, SP600125 in combination (Fig. 6B), BT- In both 474 and MCF-7 breast cancer cell lines, when LY294002 was treated in combination with chrysophanol, cell proliferation was additionally inhibited, whereas when SP600125 and SB203580 were co-treated, cell proliferation was increased again. Through these results, it was confirmed that the signaling pathway regulated by chrysophanol in ovarian cancer and breast cancer cells can actually regulate cell proliferation.

To chrysophanol Inhibiting the migration ability of ovarian cancer cells

The ability of ovarian cancer cells to migrate and invade is an important characteristic in determining the degree of metastasis of ovarian cancer. Therefore, ES2 cells and OVCAR3 cells were cultured in transwells and Ibidi culture dishes to measure changes in the migration ability of ovarian cancer cells by chrysophanol. As a result, when chrysophanol 100 μM treatment, the number of ES2 and OVCAR3 cells passing through the transwell was reduced by 69% ( p <0.001) and 52% ( p <0.001), respectively (FIG. 7A). In addition, the area between cell populations in the ibidi plate by chrysophanol was about 2 times ( p <0.01) in ES2 cells and about 1.5 times ( p <0.05) in OVCAR3 cells (Fig. 7B). These results suggest that chrysophanol can significantly reduce the migration ability of ovarian cancer cells.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

A pharmaceutical composition for preventing or treating ovarian cancer and breast cancer comprising chrysophanol as an active ingredient. The method of claim 1,
The chrysophanol is characterized in that it regulates PI3K/AKT and MAPK signaling mechanisms, a pharmaceutical composition for preventing or treating ovarian cancer and breast cancer. The method of claim 1,
A pharmaceutical composition for the prevention or treatment of ovarian cancer and breast cancer, characterized in that it further comprises a target inhibitor that inhibits the PI3K/AKT and MAPK signaling pathways. Health functional food composition for preventing or improving ovarian cancer and breast cancer comprising chrysophanol as an active ingredient. A method of inhibiting the proliferation ability of a mammalian-derived ovarian cancer cell line excluding humans or a mammalian-derived breast cancer cell line excluding humans by using a composition containing chrysophanol as an active ingredient. A method for improving the killing effect of a mammalian-derived ovarian cancer cell line excluding humans or a mammalian-derived breast cancer cell line excluding humans by using a composition containing chrysophanol as an active ingredient.

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