KR20200071384A - Pharmaceutical composition for preventing or treating gestational trophoblastic disease comprising decanoic acid - Google Patents

Abstract

The present invention relates to a composition including decanoic acid as an active ingredient. Particularly, the present invention relates to a pharmaceutical composition for preventing or treating gestational trophoblastic diseases including decanoic acid as an active ingredient, or a health-aid food composition for preventing or treating gestational trophoblastic diseases including decanoic acid as an active ingredient. The composition including decanoic acid as an active ingredient inhibits phosphorylation of downstream signal transduction molecules that control the PI3K/AKT, MAPK signal transduction mechanism in the trophoblastic cells infiltrating to the endometrium of a pregnant mother in early pregnancy, and thus inhibits proliferation and permeability of the trophoblastic cells and improves the effect of apoptosis. Therefore, the composition may be used advantageously in the field related with pharmaceuticals and health-aid foods capable of preventing and treating gestational trophoblastic diseases caused by abnormal development of the trophoblastic cells.

Description

Pharmaceutical composition for preventing or treating gestational trophoblastic disease comprising decanoic acid

The present invention relates to a composition comprising decanoic acid as an active ingredient, and more specifically, a pharmaceutical composition for preventing or treating gestational trophoblast disease comprising decanoic acid as an active ingredient and decanoic acid as an active ingredient. The present invention relates to a health functional food composition for preventing or improving gestational trophoblast disease.

Gestational trophoblastic diseases, such as chorionic cancer and moxibustion, are diseases caused by abnormal proliferation of trophoblast cells, excessive mobility and permeability. Particularly, choriocarcinoma is the most malignant disease among gestational trophoblastic diseases, and most often develops from a molar condition in early pregnancy (VCMak et al, Carcinogenesis, 2016; LDuffy et al, Journal of clinical medicine research, 2015), and trophoblastic tissue It has been reported that choriocarcinoma, a malignant tumor derived from, occurs at a rate of 1 in 20,000 to 40,000 people during pregnancy (Soper et al, Gynecol Oncol, 2004). In addition, chorionic cancer is highly vascular permeable and is known to frequently metastasize to other organs such as the lungs and vagina (Geramizadeh and Rad, Indian J Pathol Microbiol, 2012).

A common method of treating chorionic cancer is to perform a chemotherapy called EMA-CO. However, 15-25% of choriocarcinoma patients have resistance and poor prognosis for this chemotherapy (Powles et al, Br J Cancer, 2007). Therefore, in order to overcome the anti-cancer drug resistance in choriocarcinoma, an approach is needed through the development of more effective therapeutic agents.

Decanoic acid is a series of unsaturated fatty acids with 10 carbon atoms, which can be naturally extracted from coconut oil, palm oil, etc. (Dasgupta and Bhattacharyya, J. Oleo. Sci, 2009), for artificial flavors such as ice cream, fruit juice, and wine It is used as a food additive (Huang et al. , Arch. Oral Biol, 2001). In addition, decanoic acid has antiviral and antibacterial effects, and has been reported to have various biological activities when exposed to human cells (Huang et al., J. Dermatol. Sci, 2014), breast cancer, colorectal cancer, and skin cancer It is known to have anti-cancer effects in various carcinomas such as (Narayanan et al., Int. J. Mol. Sci, 2015).

As described above, various pharmacological effects by decanoic acid have been reported, but until now, there has been no known effect of decanoic acid in relation to female reproductive or pregnancy-related diseases. The effect of treating gestational trophoblastic diseases by controlling the characteristics associated with abnormal development is unknown.

Thus, the present inventors tried as a result of diligent efforts to develop a therapeutic agent for gestational trophoblast disease, confirming that decanoic acid has an effect of inhibiting trophoblast cell proliferation, migration and permeability, and apoptosis, and decanoic acid PI3K/AKT and ERK1/2 in trophoblast cells The present invention was completed by confirming that the MAPK signaling mechanism is suppressed.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating gestational trophoblastic disease comprising decanoic acid as an active ingredient.

In addition, an object of the present invention is to provide a health functional food composition for preventing or improving gestational trophoblastic disease comprising decanoic acid as an active ingredient.

In addition, it is an object of the present invention to provide a method for inhibiting the ability of the proliferation, migration and penetration of trophoblast cells using the composition.

In addition, an object of the present invention is to provide a method of improving the killing effect of trophoblast cells using the composition.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating gestational trophoblastic disease comprising decanoic acid as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving gestational trophoblast disease comprising decanoic acid as an active ingredient.

In addition, the present invention provides a method for inhibiting the proliferation, migration and penetration ability of trophoblast cells using the composition.

In addition, the present invention provides a method for improving the killing effect of trophoblast cells using the composition.

The composition comprising decanoic acid according to the present invention as an active ingredient proliferates trophoblast cells by inhibiting phosphorylation of lower signaling agents that regulate PI3K/AKT and MAPK signaling mechanisms in trophoblast cells penetrating into the endometrium of the mother in early pregnancy And as it has the effect of inhibiting the permeability and improving the cell death effect, it can be usefully used in fields related to pharmaceuticals and functional foods that can prevent and treat gestational trophoblastic diseases invented by abnormal development of trophoblast cells.

1 shows the results of analyzing the effect of decanoic acid on proliferation of trophoblast cell lines.
Figure 2 shows the results of measuring the effect of oxidative stress induced by decanoic acid treatment in trophoblast cells.
Figure 3 shows the results of analyzing the change in calcium ion concentration according to decanoic acid treatment in trophoblast cells.
Figure 4 shows the results of analyzing the effect of regulating cell proliferation according to the regulation of calcium ions in trophoblast cells.
Figure 5 shows the results of analyzing the mechanism of apoptosis when decanoic acid treatment on trophoblast cell lines.
Figure 6 shows the results of measuring the effect of inhibiting cell permeability during decanoic acid treatment on trophoblast cell lines.
Figure 7 shows the results of measuring the effect of inhibiting PI3K/AKT and ERK1/2 MAPK signaling pathways by decanoic acid treatment in trophoblast cells.
Figure 8 shows the results of analyzing the phosphorylation pattern of the signaling protein according to the combination treatment of decanoic acid, PI3K/AKT and ERK1/2 inhibitors in trophoblast cells.
9 shows the mechanism of cell proliferation inhibition and cell death by decanoic acid in trophoblast cells.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is It is well known and commonly used in the art.

In the present invention, the mechanism of cell proliferation inhibition and apoptosis in trophoblastic cells of decanoic acid was revealed (FIG. 9 ).

The present invention relates to a pharmaceutical composition for preventing or treating gestational trophoblastic disease comprising decanoic acid as an active ingredient in one aspect.

The present invention relates to a health functional food composition for preventing or improving gestational trophoblastic disease comprising decanoic acid as an active ingredient in another aspect.

As used herein, the term "composition" is considered to include any product that is made directly or indirectly by the combination of a particular component, as well as products that contain a particular component.

In the present invention, the trophoblast cell may be a mammalian-derived cell, and the mammal is a rodent (eg, mouse, rat, hamster, gerbil and guinea pig), a right head (eg, bovine) , Sheep, pigs, goats, deer, giraffes and antelopes, mechanics (e.g. horses, donkeys, rhinos and tapirs), carnivores (e.g. dogs, cats, tigers, wolves, foxes, lions, cheetahs, Leopard, raccoon, badger, puma, jaguar and wildcat), lepidoptera (rabbit and crying rabbit), carnivores (e.g. hedgehog, mole and solenoid) and primates (e.g. chimpanzee, orangutan, gorilla, Bonobono, Japanese macaque, rhesus macaque).

In the present invention, the pharmaceutical composition may be characterized by containing a pharmaceutically acceptable carrier, said carrier being an ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer substance, water, salt, Characterized by one or more selected from the group consisting of electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol and wool paper Can be.

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 by further containing any one or more adjuvants selected from the group consisting of buffers, antibacterial preservatives, surfactants, antioxidants, tonicity adjusting agents, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , It may be characterized by having a formulation selected from the group consisting of gels and powders.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as severity of symptoms, patient's weight, age, sex, mode of administration and administration time, etc., and usually, a skilled doctor is effective for the desired treatment or prevention. The dosage can be easily determined.

In the present invention, health functional food refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6622 of the Health Functional Food Act, and provides nutrients for the structure and function of the human body. Refers to foods consumed for the purpose of obtaining useful effects for health purposes such as control or physiological action.

The health functional food composition of the present invention may be formulated into a conventional health functional food formulation known in the art, granules, tablets, pills, suspensions, emulsions, syrups, gums, teas, jellies, various beverages, drinks , It may be made of an alcoholic beverage, etc., there is no particular limitation on the type of the health functional food.

The nutraceutical composition of the present invention is in the form of any medicinal herb suitable for administration to the body of an animal, including the human body, more specifically any form customary for oral administration, such as food or feed, additives or supplements of food or feed, It can be in solid form such as fortified food or feed, tablets, pills, granules, capsules and foamed formulations, or in liquid form such as solutions, suspensions, emulsions, beverages, pastes, nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, Preservatives and the like, and these components may be used independently or in combination.

According to the present invention, the decanoic acid may be characterized by inhibiting PI3K/AKT and ERK1/2 MAPK signaling mechanisms.

According to the present invention, it may be characterized in that it further comprises a target inhibitor that inhibits the PI3K/AKT or ERK1/2 MAPK signaling pathway.

According to the present invention, the gestational trophoblast disease may be characterized by at least one selected from the group consisting of choriocarcinoma, hydatidiform mole, and villous adenoma.

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

In the present invention, it may be characterized in that it further comprises etoposide, cisplatin, or paclitaxel, but is not limited thereto, and chemotherapeutic agents used as a therapeutic agent for gestational trophoblast disease, including chorionic cancer, adenoids, chorionic adenoma, etc. It can contain.

In another aspect, the present invention relates to a method for inhibiting the proliferation, migration and penetration ability of trophoblast cells using the pharmaceutical composition.

In another aspect, the present invention relates to a method of improving the killing effect of trophoblast cells using the pharmaceutical composition.

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

Example

<Experiment method>

Laboratory animals and cell culture

HTR8/SVneo cells were purchased from the American Type Culture Collection and mixed with 5% fetal bovine serum (FBS) in RPMI-1640 medium containing 2.05 mM L-Glutaimine for monolayer culture. Was used.

Experimental material

Decanoic acid, a candidate composition, was purchased from Sigma-Aldrich, Inc., and phospho-JNK, c-Jun, eIF2α, AKT, P70S6K, S6, ERK1/2 protein and IRE1α were used to confirm the signaling mechanism by decanoic acid. , cleaved Caspase-9, total-JNK, c-Jun, eIF2α, AKT, P70S6K, S6, and antibodies against ERK1/2 were purchased from Cell Signaling Techonology. In addition, antibodies to PCNA, GRP78, PERK, and TUBA were purchased from Santa Cruz Biotechnology. Ruthenium red was purchased from Abcam and BAPTA-AM was purchased from Santa Cruz Biotechnology.

BrdU Cell proliferation ability analysis

To confirm the effect of decanoic acid on the proliferative capacity of trophoblast cells, 100 µl of 5×10 ³ cells and medium cultured under FBS starvation conditions were dispensed into 96 wells, and decanoic acid was dose-dependently treated for 24 hours or 48 hours. After incubation, experiments were performed according to the manufacturer's manual using a BrdU kit (Cat No: 1167229001, Roche). After incubation, 10 μM BrdU was additionally added to each well and cultured for 2 hours in a 37° C./5% CO ₂ incubator. After labeling BrdU on chorionic cancer cells and fixing the cells, the anti-BrdU-POD solution was incubated for 90 minutes at room temperature, and then washed three times, and finally, cells with 100 μl of 3,3',5,5'-tetramethylbenzidine substrate In response, the cell proliferation ability was analyzed by measuring absorbance in 370 nm and 492 nm using an ELISA reader.

Immunofluorescence

3×10 ⁴ trophoblast cells were cultured by dispensing in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea) with 300 μl of a medium containing 5% FBS, and then additionally added to FBS starvation for 24 hours. After incubation, 400 μM of decanoic acid was treated for 24 hours, cells were fixed for 10 minutes with methanol, PCNA antibodies diluted with 2 μg/ml were treated, and mouse IgG was treated as a control group, followed by incubation at 4° C. for 16 hours. Subsequently, 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 at room temperature for 1 hour. After washing the HTR8/SVneo cells with 0.1% BSA-PBS, DAPI staining was additionally performed to simultaneously observe the target protein in the HTR8/SVneo cells as well as the nucleus. After the experiment was completed, cells were observed and photographed using a confocal microscope of LSM710 (Carl Zeiss, Thornwood, NY, USA).

Cell cycle analysis

In order to confirm the cell cycle change pattern of trophoblast cells caused by decanoic acid, 5×10 ⁵ cells were cultured in 6 wells, and further cultured in FBS starvation for 24 hours when cells were filled in a 70-80% culture dish. Next, decanoic acid was treated in a dose-dependent manner 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 0.1% BSA/PBS, fixed cells at 70% Ethanol for 16 hours, washed with 0.1% BSA/PBS, and treated under RNase A (Sigma). PI staining was performed. The stained solution was transferred to a FACS tube, and the cell cycle was analyzed by analyzing the fluorescence intensity using a flow cytometer.

DCFH Using DA Intracellular ROS Measure

To confirm the effect of decanoic acid on ROS production in HTR8/SVneo cells, it is converted to 2', 7'-dichlorofluorescin (DCF) in the presence of peroxide to fluoresce 2',7'-dichlorofluorescin diacetate (DCFH-DA, Sigma ) Was used. The HTR8/SVneo cells were detached through trypsin, and a cell pellet was obtained through centrifugation, followed by washing with PBS and incubation of 10 μM DCFH-DA in a 37° C. incubator for 30 minutes. Next, cells were washed twice with PBS and decanoic acid was dose-dependently (0, 100, 200, 400 μM) incubated in a 37° C. incubator for 1 hour. The treated cells were washed again with PBS and analyzed for DCF 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 the trophoblast cells. 3×10 ⁴ trophoblast cells were cultured by dispensing in a confocal dish with 300 μL of a medium containing 5% FBS, and then incubated with decanoic acid 400 μM in a 37° C. incubator for 2 hours. Cells were fixed with 3.7% formaldehyde and premeablized using 0.5% Triton X-100, followed by fluorescent staining with Alexa Fluor 488 Azide for 30 minutes at room temperature. Finally, after rinsing with PBS and staining DAPI, cells were observed and photographed using a confocal microscope of LSM710 (Carl Zeiss, Thornwood, NY, USA).

Intracellular Calcium ion and mitochondrial calcium ion concentration measurement

In order to confirm the effect of decanoic acid on the calcium ion concentration in HTR8/SVneo cells, 5×10 ⁵ cells were first cultured in 6 wells and further cultured in FBS starvation for 24 hours when cells were filled in 70-80% culture dishes. Thereafter, decanoic acid was treated and cultured in a 37° C./5% CO 2 incubator for 48 hours. Thereafter, cells were removed from the culture dish using trypsin, and Fluo-4 AM (Invitrogen) was incubated in a 37°C incubator for 20 minutes. The stained cells were washed once with PBS and analyzed for fluorescence intensity using a flow cytometer. The change in mitochondrial calcium ion concentration was measured using Rhod-2 AM (Cat No: R1244, Invitrogen). 5×10 ⁵ trophoblast cells were cultured in 6 wells, and further cultured in an FBS starvation state for 24 hours when the cells were filled in a 70-80% culture dish. Next, decanoic acid was treated in a dose-dependent manner and cultured in a 37° C./5% CO 2 incubator for 48 hours. Thereafter, cells were removed from the culture dish using trypsin and centrifuged to obtain a cell pellet. Cells were released in 3 μM Rhod-2 AM and incubated at 4° C. for 30 minutes. The stained cells were washed in Hank's balanced salt solution (HBSS) and analyzed for fluorescence intensity using a flow cytometer.

JC Mitochondria through -1 staining Membrane potential Measure

JC-1 mitochondrial membrane potential (MMP) changes were measured using a mitochondria staining kit (Cat No: CS0390, Sigma-Aldrich). 5×10 ⁵ trophoblast cells were cultured in 6 wells, and further cultured in an FBS starvation state for 24 hours when the cells were filled in a 70-80% culture dish. Thereafter, decanoic acid was treated in a dose-dependent manner (0, 100, 200, 400 μM) and cultured in a 37° C./5% CO ₂ incubator for 48 hours. Thereafter, cells were removed from the culture dish using trypsin and centrifuged to obtain a cell pellet. Cells were released in JC-1 staining solution and incubated in a 37° C./5% CO ₂ incubator for 20 minutes. The stained cells were centrifuged again, washed with 1x JC-1 staining buffer, and the fluorescence intensity was analyzed using a flow cytometer.

Annexin V and propidium Cell death analysis through iodide staining

In order to confirm the killing effect of trophoblast cells by decanoic acid, an experiment was conducted using FITC Annexin V cell death diagnostic kit I (BD Biosciences). First, 5×10 ⁵ cells were cultured in 6 wells, and further cultured in an FBS starvation state for 24 hours when cells were filled in a 70-80% culture dish. Thereafter, decanoic acid was treated in a dose-dependent manner (0, 100, 200, 400 μM) and cultured in a 37° C./5% CO ₂ incubator for 48 hours. Thereafter, cells were removed from the culture dish using trypsin, washed with PBS, cells were slowly mixed using 1 mL of 1× binding buffer, and centrifuged to obtain a cell pellet. Next, the cells were cultured with 200 μL of 1× binding buffer, 100 μL in a brown 1.5 mL tube, mixed with 5 μL of Annexin V and 5 μL of PI, and stained by placing the cells at room temperature for 15 minutes. Then, 400 μL of 1× binding buffer was added, and the stained solution was transferred to a 5 mL FACS tube, and the fluorescence intensity was analyzed using a flow cytometer to measure the number of dead cells.

Protein expression analysis ( Western Blot )

Proteins were quantified by Bradford protein assay (Bio-Rad, Hercules, CA, USA) after treatment with decanoic acid on trophoblast cells and extraction of whole protein from trophoblast cells. Thereafter, the extracted protein was denatured at 95°C for 5 minutes, electrophoresis was performed using a 10% SDS/PAGE gel, and then transferred to a nitrocellulose membrane, followed by incubation of the primary antibody and secondary antibody, followed by chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) reagents were used to analyze the expression of target proteins using a ChemiDoc EQ system and a Quantity One software (Bio-Rad) instrument.

cell permeability analysis

Cell permeability was 8-μm pore Transwell inserts (Corning) coated with matrigel for 2 hours at 37°C. The trophoblast cells of the medium containing 400 μM decanoic acid were incubated at 37° C. in the upper chamber for 12 hours, and then fixed with methanol for 10 minutes to measure the number of cells that penetrated downward. Thereafter, the cells were incubated for 30 minutes at room temperature with hematoxylin (Sigma), and then the cells remaining in the upper chamber were wiped off with a cotton swab. The number of infiltrated cells was measured using a DM3000 (Leica) microscope.

mRNA Expression analysis ( qPCR )

400 μM of decanoic acid was treated with trophoblast cells, and then total RNA was extracted using Trizol reagent (Invitrogen). Subsequently, cDNA was synthesized using 1 μg of RNA using AccuPower RT PreMix (Bioneer, Daejeon, Korea). Gene expression was measured using SYBR Green (Sigma) and StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). PCR conditions were incubated at 95°C for 3 minutes, and then amplified for 40 times at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 3 minutes, and normalized based on GAPDH expression.

Statistical analysis

The results of this experiment were calculated using the statistical program of SAS (statistical analysis system) and the standard error was calculated, and one-way ANOVA was performed. Significance tests were performed at the P <0.05 level.

Results and Discussion

Trophoblast Cell proliferation Decanoic acid Impact Analysis

Decanoic acid was first dose-dependently added (0, 50, 75, 100, 150, 200, 300, 400, 600, 800 μM) to analyze the changes in trophoblast cell line (HTR8/SVneo) in early pregnancy by decanoic acid After culturing for 24 hours or 48 hours in one medium, and after analyzing the cell proliferation pattern, it was confirmed that the proliferative capacity of trophoblast cells was gradually reduced by decanoic acid (FIG. 1A). When treatment with 800 μM decanoic acid was performed for 24 hours, the proliferative power of trophoblast cells decreased by 50% or more, and for 48 hours, the proliferative capacity of trophoblast cells decreased by more than 50% from 400 μM decanoic acid treatment. Subsequently, as a result of performing an immunofluorescence technique using an antibody against PCNA, which is an essential protein for DNA proliferation, the expression of PCNA is significantly reduced in the nucleus of trophoblast cells treated with 400 μM decanoic acid for 24 hours compared to the control group. It was confirmed (Fig. 1B). In addition, when treatment with decanoic acid for 48 hours, it was confirmed that the proportion of trophoblast cells in the SubG1 state increased rapidly (FIG. 1C ). In contrast, the percentage of trophoblast cells in the G2/M state tended to decrease with decanoic acid treatment. Through these results, it was confirmed that decanoic acid has an effect of inhibiting proliferation of trophoblast cells.

Trophoblast Intracellular To decane Analysis of oxidative stress induced effects

In order to confirm that oxidative stress in HTR8/SVneo cells is induced by decanoic acid, the degree of active oxygen (ROS) production in the cells was confirmed through DCF fluorescence observation. As a result, DCF fluorescence in HTR8/SVneo cells treated with decanoic acid was dose-dependently increased (FIG. 2A ). When treated with 400 μM decanoic acid, the amount of free radical production increased by about 22.3 times compared to the control group. In addition, as a result of confirming the degree of lipid peroxidation induced by free radicals, it was confirmed that lipid peroxidation was rapidly increased in trophoblast cells treated with 400 μM decanoic acid for 2 hours compared to the control group, and when it was quantified, it showed an increase rate of about 2.5 times. 2B).

Next, intracellular calcium ion concentration and mitochondrial specific calcium ion concentration were analyzed by flow cytometry through Fluo-4 staining and Rhod-2 staining, respectively. It was confirmed that the concentration of mitochondrial specific calcium ion increased by about 2.9-fold with 48 μM decanoic acid treatment (FIGS. 3A and 3B ). Next, BAPTA-AM, a calcium ion channel inhibitor Ruthenium Red (8 μM) and a calcium ion scavenger, is used to analyze whether an increase in mitochondrial specific calcium ions in trophoblast cells by decanoic acid is directly involved in proliferation of trophoblast cells. As a result of treating (1 μM) with decanoic acid, it was confirmed that the mitochondrial calcium ion concentration increased by decanoic acid during BAPTA-AM treatment was significantly decreased (FIG. 4A ). In fact, the proliferation of trophoblast cells was confirmed after treatment with decanoic acid and Ruthenium Red and BAPTA-AM. As a result, there was no significant change in cell proliferation by Ruthenium Red, but decreased cell proliferation by decanoic acid by BAPTA-AM treatment. It was found to recover (Figure 4B). Through these results, it was confirmed that mitochondrial specific calcium influx into the trophoblast cell is mediated by oxidative stress by decanoic acid in the trophoblast cell, which is closely related to the proliferation of trophoblast cells.

Trophoblast Intracellular To decane Analysis of cell death mechanism

After 6, 24, 48 hours incubation of decanoic acid in a dose-dependent manner (0, 100, 200, 400 μM) to confirm whether the mitochondrial membrane potential changes according to the calcium ion over-introduction into the mitochondrial in the trophoblast cell Mitochondrial membrane potential was measured by JC-1 staining. As a result, it was confirmed that the mitochondrial membrane potential in the trophoblast cell was decreased by decanoic acid in a dose and time-dependent manner (FIG. 5A ). Upon treatment for 6 hours and 24 hours and 48 hours, mitochondrial membrane potentials of 1.4 times, 3.0 times, and 7.3 times compared to the control group, respectively, were confirmed.

In addition, in order to analyze the effect of decanoic acid on the apoptotic cells, the cell death was carried out after 48 hours of incubation with decanoic acid in the trophoblast cells in a dose-dependent manner (0, 100, 200, 400 μM), followed by Annexin V and PI staining. The number of trophoblast cells that occurred was measured using FACS. As a result, it was confirmed that cell death increased in a dose-dependent manner when treated with decanoic acid compared to the control group (FIG. 5B ). In response to 400 μM decanoic acid, about 33.9% of the trophoblast cells were present in a dead state, which was about 5.8 times that of the control group.

JNK protein is a signaling protein that affects mitochondria and vesicles and is activated by ROS. To confirm that vesicular stress is induced by decanoic acid, phosphorylation of JNK, a signaling protein closely related to endoplasmic reticulum stress, and c-Jun, a sub-signaling protein, was examined. As a result, it was confirmed that phosphorylation was increased by 400 μM decanoic acid in both JNK and c-Jun, and that expression and activity of vesicle stress-related proteins such as GRP78, PERK, eIF2α, and IRE1α increased (FIG. 5C). GRP78 protein is a chaperone protein targeted for vesicle stress, and PERK and IRE1α are vesicle stress sensing proteins. In addition, eIF2α is well known as a sub-protein phosphorylated by PERK. In addition, the cleaved form of Caspase-9, a representative protein of the mitochondrial-dependent apoptosis pathway, also showed a tendency to increase by decanoic acid.

Through these results, it was confirmed that decanoic acid induces cell death by mediating endoplasmic reticulum stress in the trophoblast cells.

Trophoblast Cellular Permeability Decanoic acid Impact Analysis

To confirm the effect of decanoic acid on the permeability of trophoblast cells, HTR8/SVneo cells were dispensed into a transwell coated with Matrigel and incubated for 12 hours in a medium containing decanoic acid (400 μM). As a result, permeability of trophoblast cells was reduced by about 66% by decanoic acid (FIG. 6A). In addition, as a result of analyzing the mRNA level expression of a protein that improves the permeability of cells by regulating the extracellular matrix such as MMP2, MMP14, PLAU, FOXM1, the expression of the corresponding genes was significantly reduced compared to the control group when treated with decanoic acid (Fig. 6B-6E). Through these results, it was confirmed that decanoic acid can suppress excessive permeability of trophoblast cells.

PI3K / With AKT ERK1 /2 in the signaling pathway Decanoic acid Impact Analysis

To confirm the signaling mechanism affecting cell proliferation inhibition and cell death induced by decanoic acid in trophoblastic cells, the phosphorylation of ERK1/2 MAPK and AKT associated with proliferation was dose-dependently decanoic acid (FIG. 7). Analysis was done using blots. As a result, it was confirmed that decanoic acid not only inhibits phosphorylation of proteins contained in PI3K/AKT signaling pathways such as AKT, P70S6K, S6 in trophoblast cells, but also inhibits the activity of ERK1/2 protein.

In addition, the activity of AKT, P70S6K, S6, and ERK1/2 signaling proteins was treated by treating with a specific inhibitor for AKT LY294002 (20 μM) and a specific inhibitor for ERK1/2 U0126 (20 μM) in combination with decanoic acid. Was analyzed by Western blot (Fig. 8). As a result, it was found that the PI3K/AKT signaling pathways including P70S6K and S6 increased activity with U0126 combination treatment, while the activity of ERK1/2 protein was further reduced by treatment with LY294002. Through these results, the signaling pathway regulated by decanoic acid suggests that the AKT signaling protein acts higher than the ERK1/2 protein and has a cross effect.

Since specific parts of the present invention have been described in detail above, it is obvious that for those skilled in the art, this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

A pharmaceutical composition for the prevention or treatment of gestational trophoblastic disease comprising decanoic acid as an active ingredient. According to claim 1,
The decanoic acid PI3K / AKT and ERK1 / 2 MAPK signaling pharmaceutical composition for preventing or treating gestational trophoblast disease characterized in that it inhibits the signaling mechanism. According to claim 1,
A pharmaceutical composition for preventing or treating gestational trophoblastic disease, further comprising a target inhibitor that inhibits the PI3K/AKT or ERK1/2 MAPK signaling pathway. According to claim 1,
The gestational trophoblastic disease is a pharmaceutical composition for preventing or treating gestational trophoblastic disease, characterized in that at least one selected from the group consisting of choriocarcinoma, hydatidiform mole, and villous adenoma. A health functional food composition for preventing or improving gestational trophoblast disease, comprising decanoic acid as an active ingredient. A method for inhibiting the proliferation, migration and penetration ability of trophoblast cells using the pharmaceutical composition for preventing or treating gestational trophoblast disease according to claim 1. A method for improving the killing effect of trophoblast cells using the pharmaceutical composition for preventing or treating gestational trophoblast disease according to claim 1.

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