KR20230085385A - Pharmaceutical composition for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblast cells comprising bifenthrin - Google Patents

Abstract

The present invention relates to a composition comprising bifenthrin as an active ingredient and, more specifically, to a pharmaceutical composition comprising bifenthrin as an active ingredient for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblast cells, and a functional health food composition comprising bifenthrin as an active ingredient for preventing or alleviating diseases related to abnormal proliferation of endometrial cells or trophoblast cells. The composition comprising bifenthrin as an active ingredient according to the present invention regulates the phosphorylation of PI3K/AKT and MAPK cell transduction mechanisms and their downstream signaling substances in endometrial cells or trophoblast cells in various ways and increases the expression of endoplasmic reticulum stress response factors involved in oxidative stress regulation to suppress the proliferation of endometrial cells and trophoblast cells and induce cell death, so the composition can be advantageously used in the fields related to medicines and functional foods that can prevent and treat diseases related to abnormal proliferation of endometrial cells or trophoblast cells.

Description

Pharmaceutical composition for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblast cells comprising bifenthrin

The present invention relates to a composition containing bifenthrin as an active ingredient, and more particularly, to a pharmaceutical composition for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblast cells containing bifenthrin as an active ingredient; and a health functional food composition for preventing or improving abnormal proliferation-related diseases of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.

Diseases such as endometriosis, endometrial hyperplasia, endometrial polyp, and endometrial cancer are diseases caused by abnormal proliferation of endometrial cells. Among them, endometriosis is a disease in which endometrial cells abnormally proliferate in the ovaries, fallopian tubes, and abdominal wall of women of childbearing age, causing infertility in 35-50% of patients (Vigano et al, Best Pract Res Clin Obstet Gynaecol, 2004; Bulun , N Engl J Med, 2009), and studies on the pathological mechanism thereof are being actively conducted, but the exact cause of the disease has not yet been identified. The basic treatment methods for endometriosis include surgical removal and hormonal therapy, and because endometriosis is diagnosed based on symptoms rather than prognostic factors, the recurrence rate within 5 years is low even after surgical removal. is known to approach 30-40% (Busacca et al., J Obstet Gynecol, 2006: Vignali et al., J Minim Invasive Gynecol, 2005). In addition, endometrial cancer is a malignant tumor caused by overgrowth of uterine tissue, and its incidence is continuously increasing due to the increase in the average age of childbirth and changes in lifestyle, and is known as a gynecological tumor with the highest incidence in the United States (Mills , A. M. and T. A. Longacre, Semin Diagn Pathol, 2010).

On the other hand, gestational trophoblast diseases such as chorionic carcinoma, molar molars, and choriocarcinoma are diseases caused by abnormal proliferation, excessive mobility and permeability of trophoblast cells. In particular, chorionic carcinoma is the most malignant disease among gestational trophoblast diseases, and most cases arise from molar follicles in the early stages of pregnancy (VCMak et al, Carcinogenesis, 2016; LDuffy et al, Journal of clinical medicine research, 2015), and trophoblast tissue Choriocarcinoma, a malignant tumor derived from , has been reported to occur at a rate of 1 per 20,000 to 40,000 during pregnancy (Soper et al, Gynecol Oncol, 2004). In addition, it is known that choriocarcinoma has very high vascular permeability and metastases to other organs such as the lung or vagina frequently occur (Geramizadeh and Rad, Indian J Pathol Microbiol, 2012). A common treatment for choriocarcinoma is to administer chemotherapy called EMA-CO. However, 15-25% of choriocarcinoma patients are resistant to these chemotherapy and show poor prognosis (Powles et al, Br J Cancer, 2007). Therefore, in order to overcome anticancer drug resistance in choriocarcinoma, there is a need for an approach through the development of more effective therapeutic agents.

As such, abnormal proliferation of endometrial cells or trophoblast cells causes various serious diseases, and all of these diseases require the development of new therapeutic agents due to the limitations of conventional treatments.

Under the aforementioned technical background, the present inventors have made diligent efforts to develop new substances for the treatment of abnormal proliferation-related diseases of endometrial cells or trophoblast cells. The present invention was completed by confirming that proliferation, migration, and invasiveness of endometrial cells or trophoblast cells were inhibited, and cell death was induced by regulating the activity of the MAPK signaling pathway.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases associated with abnormal proliferation of endometrial cells or trophoblasts, containing bifenthrin as an active ingredient.

In addition, an object of the present invention is to provide a health functional food composition for preventing or improving diseases related to abnormal proliferation of endometrial cells or trophoblast cells, containing bifenthrin as an active ingredient.

In addition, an object of the present invention is to provide a method for inhibiting proliferation, migration and penetration of endometrial cells or trophoblasts by using a composition containing bifenthrin as an active ingredient.

In addition, an object of the present invention is to provide a method for enhancing the killing effect of endometrial cells or trophoblast cells by using a composition containing bifenthrin as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating diseases associated with abnormal proliferation of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving diseases related to abnormal proliferation of endometrial cells or trophoblasts, containing bifenthrin as an active ingredient.

In addition, the present invention provides a method for inhibiting proliferation, migration and penetration of endometrial cells or trophoblasts by using a composition containing bifenthrin as an active ingredient.

In addition, the present invention provides a method for enhancing the killing effect of endometrial cells or trophoblast cells by using a composition containing bifenthrin as an active ingredient.

The composition containing bifenthrin as an active ingredient according to the present invention variously regulates the phosphorylation of PI3K/AKT and MAPK cell transduction mechanisms and sub-signal transmitters in endometrial cells or trophoblast cells, and is involved in oxidative stress regulation. By increasing the expression of endoplasmic reticulum stress response factors, it inhibits proliferation of endometrial cells and trophoblast cells and induces apoptosis, thereby preventing and treating diseases associated with abnormal proliferation of endometrial cells or trophoblast cells. And it can be usefully used in fields related to functional foods.

1 shows the results of analyzing the effect of bifenthrin on the proliferation of porcine trophoblast and endometrial cells.
Figure 2 shows the results of analyzing the effect of inducing death of porcine trophoblast and endometrial cells by bifenthrin.
Figure 3 shows the results of analyzing the cell cycle progression inhibition effect of porcine trophoblast cells and endometrial cells by bifenthrin.
Figure 4 shows the results of analyzing the effect of inducing mitochondrial dysfunction in porcine trophoblast cells and endometrial cells by bifenthrin.
5 shows the results of analyzing the effects of bifenthrin on inducing endoplasmic reticulum stress and inhibiting calcium homeostasis in porcine trophoblast cells and endometrial cells.
Figure 6 shows the results of analyzing the effect of bifenthrin on signal transduction mechanisms in porcine trophoblast cells and endometrial cells.
Figure 7 shows the results of analyzing the effect of bifenthrin on spheroid formation and growth inhibition of porcine trophoblast and endometrial cells.
8 shows the mechanism of inhibition of cell proliferation and cell death by bifenthrin in trophoblast or endometrial cells.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In the present invention, the mechanism of bifenthrin's inhibition of trophoblast or endometrial cell proliferation and apoptosis was found (FIG. 8).

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating diseases associated with abnormal proliferation of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.

In another aspect, the present invention relates to a functional health food composition for preventing or improving diseases related to abnormal proliferation of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient.

As used herein, the term "composition" is considered to include not only products containing specific components, but also any products made directly or indirectly by combining specific components.

In the present invention, the trophoblast cells or endometrial epithelial cells may be mammalian-derived cells, and the mammals are rodents (eg, mice, rats, hamsters, gerbils and guinea pigs), corticosteroids ( e.g. cattle, sheep, pigs, goats, deer, giraffes and antelopes), branch orders (e.g. horses, donkeys, rhinos and tapirs), carnivores (e.g. dogs, cats, tigers, wolves, foxes) , lions, cheetahs, leopards, raccoons, badgers, pumas, jaguars and bobcats), lagomorphs (rabbits and songbirds), insectivores (e.g. hedgehogs, moles and solenodons) and primates (e.g. chimpanzees, orangutans, gorillas, bonobos, Japanese macaques, rhesus monkeys).

In the present invention, the pharmaceutical composition may be characterized by containing a pharmaceutically acceptable carrier, and the carrier may include ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer substances, water, salt, At least one 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. can

In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, buccal, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration. It may be characterized in that it further contains at least one adjuvant selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity adjusters, 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.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the severity of symptoms, the patient's weight, age, sex, administration method and administration time, and usually a skilled physician is effective in the treatment or prevention desired. 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 useful functionalities for the human body according to Act No. It refers to food consumed for the purpose of obtaining useful effects for health purposes such as regulation or physiological action.

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

The health functional food composition of the present invention is any herbal form suitable for administration to the human body or animal body, more specifically, any form conventional for oral administration, such as food or feed, food or feed additives and supplements, Solid forms such as fortified foods or feeds, tablets, pills, granules, capsules and effervescent formulations, or liquid forms such as solutions, suspensions, emulsions, beverages, pastes, etc., nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, It may contain preservatives and the like, and these components may be used independently or in combination.

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

According to the present invention, any disease associated with abnormal proliferation of endometrial cells or trophoblast cells is possible as long as it is caused by abnormal development of endometrial cells or trophoblast cells. For example, the disease associated with abnormal proliferation of endometrial cells is 1 selected from the group consisting of endometriosis, endometrial hyperplasia, endometrial polyp, and endometrial cancer. There may be more than one species. In addition, the abnormal proliferation-related disease of trophoblastic cells may be a gestational trophoblastic disease, and the gestational trophoblastic disease is a group consisting of choriocarcinoma, hydatidiform mole, and villous adenoma. It may be one or more selected from.

In the present invention, the bifenthrin may increase the expression of endoplasmic reticulum stress response factors involved in oxidative stress regulation in trophoblast and endometrial cells.

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

In another aspect, the present invention relates to a method for inhibiting proliferation, migration, and invasion of endometrial cells or trophoblast cells derived from non-human mammals by using a composition containing bifenthrin as an active ingredient.

In another aspect, the present invention relates to a method for enhancing the killing effect of non-human endometrial cells or trophoblast cells by using a composition containing bifenthrin as an active ingredient.

[Example]

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

Experiment method

cell culture

The porcine trophoblast cell line (pTr) was extracted from the blastocyst of a sow on day 12 of pregnancy, and the porcine endometrial cell line (pLE) was extracted from the endometrium of a sow on day 12 of pregnancy. For monolayer culture of the two cells, they were cultured in DMEM/F12 1:1 medium supplemented with 10% fetal bovine serum, 1% antibiotics and 0.1% insulin.

experimental material

Bifnthrin was purchased and used from ChemService, and phospho-AKT, ERK1/2, P70S6K, S6, JNK, P38, eIF2α proteins and total-AKT, ERK1/2, P70S6K, S6, JNK, P38, BAX, BAK, and Cytochrome C proteins were purchased from Cell Signaling Technology. In addition, GRP78, α-tubulin, phospho-PERK, and PCNA antibodies were purchased from Santa Cruz Biotechnology. In addition, to investigate the effect of inhibition of the target signaling pathway, PI3K inhibitor LY294002 was purchased from Cell Signaling Technology, and ERK1/2, JNK, and P38 inhibitors U0126, SP600125, and SB203580 were purchased from Enzo Life Science and used. .

tetrazolium Analysis of cytotoxic effect using bromide

In order to evaluate the cytotoxicity of Bifenthrin, pTr and pLE cells cultured in 96 well were treated with Bifenthrin and cultured for 48 hours. Then, 10 μL of MTT labeling reagent was additionally dispensed and incubated at 37° C. for 4 hours. After dispensing 100 μL of solubilization solution into each well and incubating in an incubator at 37℃/5% CO ₂ for 16 hours, cell proliferation ability was analyzed by measuring absorbance at 560 nm and 650 nm using an ELISA reader.

Trypan Analysis of cell proliferation ability using blue staining

In order to confirm the proliferation inhibitory effect of bifenthrin, pTr and pLE cell lines were cultured for 24 hours, 48 hours, and 72 hours in media treated with bifenthrin (0, 2, 5, 10 μM) in a dose-dependent manner. Thereafter, the cells were removed from the culture dish using 0.05% trypsin and then stained using Trypan blue. After inoculating 20 μL of the stained cells in a hematocytometer and counting the number of live and dead cells, the total number of cells was calculated through the following equation. (number of cells counted) / 4 × (dilution factor) × (volume of cell suspension). In addition, cell viability was calculated as (number of living cells) / (total number of cells) × 100, and the experiment was repeated three times.

Transwell Cell migration ability analysis using

pTr cells were treated with 10 μM bifenthrin or DMSO (control) and cultured for 14 hours in Transwell inserts (catalog number: 3422, Corning, Corning, NY, USA). Thereafter, the cells were fixed with methanol for 10 minutes and then stained with hematoxylin for 30 minutes at room temperature. After staining, the remaining hematoxylin was removed using primary water and the membrane was separated using a single blade. Membrane was fixed to the slide through mounting and then photographed using an optical microscope DM3000 (Leica, Wetzlar, Germany).

Annexin with V Propidium Apoptosis analysis by iodide (PI) staining

In order to confirm the apoptosis effect by bifenthrin, an experiment was performed using FITC Annexin V Cell Death Diagnostic Kit I (BD Biosciences). In the case of pTr and pLE cells, 1 x 10 ⁵ cells were cultured in a 6-well culture dish and treated with bifenthrin (0, 2, 5, 10 μM) for 48 hours. Thereafter, the cells were removed from the culture dish using trypsin and washed with PBS. Cells obtained by centrifugation were cultured in suspension in 1x Annexin binding buffer, and 5 μL each of Annexin V and PI were added and stained for 15 minutes. Fluorescent signals were measured using a flow cytometer.

immunofluorescence method cell proliferation using marker's Expression analysis

After culturing pTr and pLE cells in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea), they were treated with 10 μM of bifenthrin for 24 hours. After fixing the cells with methanol, they were treated with PCNA antibody and incubated at 4°C for 16 hours. Subsequently, as a secondary antibody, goat anti-mouse IgG Alexa 488 (catalog number: A-11001, Invitrogen, Carlsbad, CA, USA) was diluted 1:200 in antibody dilution buffer and reacted at room temperature for 1 hour. After washing with 0.1% BSA-PBS to remove unbound secondary antibodies, the nuclei were stained with DAPI. Fluorescent signals were captured using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope.

Analysis of cell cycle progression via PI staining

For cell cycle analysis, the amount of DNA was measured by propidium iodide staining. The pTr and pLE cells treated with bifenthrin (0, 2, 5, and 10 μM) for 48 hours were detached from the culture dish using trypsin, and then fixed using ethanol for 16 hours. The fixed cells were stained with RNase A (Sigma) and PI (BD Biosciences), and fluorescence signals were measured through flow cytometry. Then, the ratio of each cell cycle was analyzed through PI signal quantification.

DCFH -Intracellular active oxygen analysis using DA staining

Intracellular active oxygen analysis was conducted through DCFH-DA staining. First, pTr and pLE cells cultured in a 100π culture dish were removed, and then incubated with DCFH-DA (Sigma) for 30 minutes to infiltrate the cells. Thereafter, the pTr and pLE cells were treated with Bifenthrin (0, 2, 5, 10 μM) for 1 hour. In addition, to verify the recovery of active oxygen levels, a scavenger, N-acetyl-1-glutamine (NAC), was co-treated. Finally, the fluorescence signal of DCFH-DA was measured using a flow cytometer.

JC Mitochondria using -1 staining membrane potential analyze

Mitochondrial membrane potential was measured using a Mitochondrial staining kit (Sigma). The pTr and pLE cells treated with bifenthrin (0, 2, 5, and 10 μM) for 48 hours were removed from the culture dish and centrifuged to obtain a cell pellet. Thereafter, the cells were incubated in JC-1 staining solution at 37° C. for 20 minutes, and then washed using JC-1 staining buffer. Thereafter, the fluorescence signal of JC-1 in cells was detected using a flow cytometer.

Fluo Analysis of cytosolic calcium concentration using -4 staining

In order to confirm the effect of Bifenthrin on intracellular Ca ²⁺ concentration, pTr and pLE cells ^cultured in 6 wells were dose-dependently treated with Bifenthrin (0, 25, 50, 100 μM) and cultured for 48 hours. did Thereafter, cells were detached from the culture dish using trypsin and incubated with Fluo-4 AM (Invitrogen) for 20 minutes. The stained cells were washed with PBS, and fluorescence signals were analyzed using a flow cytometer.

Rhod Analysis of mitochondrial calcium concentration using -2 staining

In order to detect the level of mitochondrial calcium, an experiment was conducted using Rhod-2 AM fluorescent protein that passes through mitochondria and binds to calcium ions inside. The bifenthrin-treated pTr and pLE cells were stained with Rhod-2 AM and then washed with HBSS medium to remove unbound Rhod-2 AM. The stained cells were incubated in HBSS medium without Ca ^{2 +} Mg ²⁺ for 10 minutes at 37° C., ^and analysis was performed. The fluorescence intensity of Rhod-2 AM was measured using a flow cytometer.

western blot Protein expression level analysis using

Total protein was extracted from bifenthrin-treated pTr and pLE cells, and protein concentration was measured by Bradford protein assay. Thereafter, the extracted proteins were denatured at 95° C. for 5 minutes, and electrophoresis was performed on an SDS/PAGE gel. After transferring this to a nitrocellulose membrane, the primary antibody and the secondary antibody were sequentially incubated. Antibodies attached to the membrane were reacted using chemiluminescenc detection (SuperSignal West Pico, Pierce) reagent, and protein expression was analyzed using the ChemiDoc EQ system.

statistical analysis

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

Results and Discussion

to bifenthrin Pig by Trophoblast Cell and endometrial cell proliferation inhibitory effect

To confirm the effect of bifenthrin on the proliferation of trophoblast and endometrial cells, bifenthrin was dose-dependently (0, 1, 2, 5, 10, 20, 30, 50 μM), changes in cell viability were confirmed. As a result, it was confirmed that the proliferative capacity of pTr and pLE cells decreased by up to 30% as the concentration of bifenthrin increased (FIG. 1). Through this, it was confirmed that bifenthrin can significantly reduce the proliferation of trophoblast and endometrial cells.

to bifenthrin Pig by trophoblast cells and endometrial cells cell death effect

Next, in order to confirm the effect of bifenthrin according to the treatment time, pTr and pLE cells were treated with bifenthrin (0, 2, 5, and 10 μM), and trypan blue staining was performed 24, 48, and 72 hours later. As a result, it was confirmed that the cell proliferation rate decreased over time in bifenthrin-treated cells compared to the control group (FIG. 2A). Additionally, as a result of analyzing the ratio of viable cells to the total number of cells, it was confirmed that the slope of the decrease in viability according to the concentration significantly increased as the treatment time of Bifenthrin increased (FIG. 2B). In addition, as a result of confirming the apoptosis inducing effect of bifenthrin through Annexin V experiments, it was confirmed that both early and late apoptosis significantly increased in pTr and pLE cells (FIG. 2C, D). Through these results, it was confirmed that bifenthrin has an effect of inhibiting cell proliferation and inducing cell death in trophoblast and endometrial cells.

to bifenthrin Pig by trophoblast cells and endometrial cells cell cycle inhibitory effect

Next, to verify the cell proliferation inhibitory effect of Bifenthrin at the molecular level, we investigated changes in the expression of PCNA, an essential protein for cell proliferation. was found (Fig. 3A, B). In addition, as a result of cell cycle analysis through PI staining, it was confirmed that bifenthrin treatment increased the ratio of sub-G1 phase cells by about 4-fold or more in pTr and pLE cells (Fig. 3C, D). In addition, through gene expression tests of cyclins required for cell cycle progression, it was confirmed that bifenthrin's cell cycle inhibition was induced by reducing CCND1 and CCNE1 (FIG. 3E).

to bifenthrin Pig by trophoblast cells and endometrial cells Disruption of mitochondrial homeostasis and consequent generation of reactive oxygen species

JC-1 assay was performed to confirm the effect of bifenthrin on mitochondrial homeostasis. As a result, the signal of JC-1, which is an indicator of mitochondrial depolarization, increased 5.5-fold and 3.8-fold, respectively, in pTr and pLE cells according to bifenthrin treatment (FIG. 4A, B). Since mitochondria are a major source of reactive oxygen species, DCFH-DA staining was performed to determine whether reactive oxygen species were generated in endometrial cells following bifenthrin treatment. As expected, Bifenthrin increased intracellular ROS by about 1.5-fold in pTr and pLE, and it was confirmed that intracellular ROS levels were restored when treated together with NAC, known as an ROS inhibitor (FIG. 4C, D). Additionally, as a result of confirming lipid peroxidation in pTr and pLE cells to verify that reactive oxygen species generated by bifenthrin actually induce cell damage, green fluorescence signals detecting products of lipid oxidation increased when bifenthrin was treated. was confirmed (Fig. 4E, F). Through these results, it was confirmed that bifenthrin induces the generation of reactive oxygen species by inducing mitochondrial depolarization in porcine trophoblast and endometrial cells.

trophoblast cells and to endometrial cells working of bifenthrin Analysis of endoplasmic reticulum stress induction and calcium regulation inhibitory effect

Considering that bifenthrin generates intracellular reactive oxygen species and impairs mitochondrial function, it was expected to induce endoplasmic reticulum stress. To verify this, the expression of signaling proteins involved in the stress response in the endoplasmic reticulum was measured by Western blotting. As a result, as the concentration of bifenthrin increased, the expression of GRP78, a representative chaperone protein, increased significantly, and the expression of both phsophorylated-eIF2α, a component of the translation initiation complex, and the transcription factor GADD153 increased. (Fig. 5A, B). In addition, since damage to endoplasmic reticulum function has a great effect on intracellular calcium ion regulation, changes in intracellular calcium were analyzed using Rhod2-AM and Fluo4-AM staining. First, as a result of analyzing the fluorescence signal of Rhod2-AM through flow cytometry, it was confirmed that bifenthrin increased mitochondrial calcium in pTr and pLE cells by about 160% and 180% (Fig. 5C, D). In addition, through the results of Fluo4-AM analysis, it was confirmed that cytoplasmic calcium increased by 110% and 150%, respectively (FIG. 5E, F). Through these results, it was confirmed that bifenthrin inhibits the ability to regulate intracellular calcium homeostasis by inducing the endoplasmic reticulum stress response.

pig's trophoblast cells and to endometrial cells working of bifenthrin Signal transduction pathway regulation effect analysis

Under the assumption that the above-identified intracellular stress response and homeostasis disruption would affect the intracellular survival signaling pathway, protein activation patterns of the PI3K and MAPK pathways were investigated through Western blot analysis. As a result, AKT, p70S6K, S6, ERK1/2, and JNK were detected in pTr cells according to the treatment concentration of Bifenthrin. Phosphorylation of p38 protein was all significantly increased (Fig. 6A, C). In addition, the MAPK pathway was increased in pLE cells (FIG. 6B), and the phosphorylation of AKT and p70S6K in the PI3K pathway decreased with bifenthrin treatment. Phosphorylation was slightly increased (Fig. 6D). Through these results, it was confirmed that bifenthrin activated the MAPK and PI3K signaling pathways in porcine trophoblast cells, and activated the MAPK pathway and inhibited the PI3K pathway in endometrial cells.

to bifenthrin of pigs by trophoblast cells and endometrial cells spheroid formation and growth inhibitory effects

Spheroid cell culture was performed to determine whether bifenthrin also inhibited the viability of 3D cultured cells, not adherent culture. As a result, 10 μM of bifenthrin inhibited the proliferation of pTr and pLE cells and reduced the growth of spheroid cells (Fig. 7A). In addition, as a result of analyzing the colony number and total area of pTr and pLE cell spheroids using the Image J program, the number of colonies decreased by 60% and 73%, respectively, and the size decreased by 75% and 80%, respectively, according to bifenthrin treatment. % was confirmed (Fig. 7B, C). Through these results, it was confirmed that bifenthrin had an effect of significantly inhibiting cell proliferation in porcine trophoblast and endometrial cells forming a 3D structure.

Having described specific parts of the present invention in detail above, it is clear that these specific descriptions are only preferred embodiments for those skilled in the art, 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 (8)

A pharmaceutical composition for preventing or treating diseases related to abnormal proliferation of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient. According to claim 1,
The disease associated with abnormal proliferation of endometrial cells is at least one selected from the group consisting of endometriosis, endometrial hyperplasia, endometrial polyp, and endometrial cancer. A pharmaceutical composition for preventing or treating abnormal proliferation-related diseases of endometrial cells or trophoblast cells. According to claim 1,
A pharmaceutical composition for preventing or treating abnormal proliferation-related diseases of endometrial cells or trophoblastic cells, characterized in that the abnormal proliferation-related disease of trophoblastic cells is gestational trophoblastic disease. According to claim 3,
Prevention of diseases associated with abnormal proliferation of endometrial cells or trophoblast cells, characterized in that the gestational trophoblast disease is at least one selected from the group consisting of choriocarcinoma, hydatidiform mole, and villous adenoma. or a pharmaceutical composition for treatment. According to claim 1,
A pharmaceutical composition for preventing or treating abnormal proliferation-related diseases of endometrial cells or trophoblast cells, characterized in that the bifenthrin regulates PI3K/AKT and MAPK signaling mechanisms. A health functional food composition for preventing or improving abnormal proliferation-related diseases of endometrial cells or trophoblasts, comprising bifenthrin as an active ingredient. A method of inhibiting proliferation, migration and invasion of endometrial cells or trophoblasts derived from non-human mammals by using a composition containing bifenthrin as an active ingredient. A method for enhancing the killing effect of endometrial cells or trophoblast cells derived from non-human mammals by using a composition containing bifenthrin as an active ingredient.

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