KR101505175B1 - A novel composition for supressing metastasis of cancer - Google Patents

Abstract

The present invention relates to a novel composition for suppressing cancer metastasis, and more particularly to a composition for inhibiting tumor metastasis comprising a flavonoid effective ingredient.

Description

[0001] The present invention relates to a novel composition for suppressing metastasis of cancer,

The present invention relates to a novel composition for suppressing cancer metastasis, and more particularly to a composition for inhibiting tumor metastasis comprising a flavonoid effective ingredient.

The number of cancer deaths in Korea in 2002 was 62,887 out of 246,515 deaths in Korea (29.6% of male deaths and 20.5% of female deaths), accounting for 25.5% have. This means that about 131 people die per 100,000 population in Korea (2002 National Statistical Office).

From the viewpoint of cancer treatment, one of the major reasons for not decreasing the mortality rate of cancer patients is that they do not inhibit the invasion and metastasis of cancer cells during malignant tumorization.

Recent studies have identified genes associated with metastasis (eg, Twist, KAI-1), and thus identify molecular mechanisms of metastasis.

Therefore, no drugs or treatment methods have been reported that can effectively inhibit or prevent cancer metastasis.

A recent statistical analysis in the United States shows that metastatic cancer did not improve survival over the last 20-30 years for 5 years. Therefore, in the United States, cancer treatment strategies have been set to increase cancer cure rate by inhibiting cancer metastasis.

Therefore, the understanding of the molecular mechanisms of metastasis and invasion, which are characteristic of malignant tumors, will ultimately contribute to the survival rate of cancer patients by providing a foundation for the prevention and treatment of cancer metastasis.

EMT (epithelial-mesenchymal transition) in malignant tumors represents a series of processes in which epithelial cells are transformed into mesenchymal cells, such as fibroblasts (Kang Y, Massague J., epithelial-mesenchymal transitions: twist in development and metastasis (2004) Cell vol. 118 (3), 277-279)

These EMTs are observed in embryonic cell layer movement and organogenesis in normal embryonic development stages (Nakaya Y, Sheng G., EMT in developmental morphogenesis. Cancer Letters (2013) vol. 341 (1), 9-15).

In the 1990s, EMT was reported to be a pathophysiologic factor mediating tumor malignancy and chronic fibrosis processes (Hay et al., An overview of epithelio-mesenchymal transformation, Acta. 1), 8-20).

In the course of tumor progression, EMT is specifically involved in tumor invasion, intravasation and extravasation of metastatic cancer cells, and in the case of chronic fibrosis, EMT plays an important role in the production of fibroblasts that lead to accumulation of extracellular matrix (Yilmaz M, Christofori G., EMT, the cytoskeleton, and cancer cell invasion (2009) Cancer Metastasis Rev. vol. 28 (1-2) 15-33).

The most important hallmark of the EMT process is the deformation of the cell-cell junctions (tight junctions, desmosomes, adherence junctions, gap junctions) between epithelial cells, resulting in destruction of epithelial polarity and rearrangement of the actin cytoskeleton. These changes in the cytoskeleton promote cell migration and turn into spindle-shaped mesenchymal cells. Therefore, understanding of the EMT phenomenon at the molecular level, which is one of the characteristics of cancer cells in malignant tumorigenesis, will broaden the understanding of EMT-related diseases and contribute to finding targets for artificially modulating EMT (Yilmaz M, Christofori G., EMT, the cytoskeleton, and cancer cell invasion (2009) Cancer Metastasis Rev. vol. 28 (1-2) 15-33).

Transforming growth factor-beta (TGF-beta) is expressed in most cells in the body and plays an essential role in inhibition of cell growth, cell differentiation and cell death (Shi Y, Massague J., Mechani, TGF-beta signaling cell membrane to the nucleus (2003) Cell vol. 113 (6) 685-700).

However, recent studies have shown that TGF-beta has both tumor suppressor function that inhibits epithelial cell growth or cell death, as well as tumor promoting function that contributes to tumor malignancy (Wakefield LM, Roberts AB., TGF-beta signaling: positive and negative effects on tumorigenesis. Curr Opin. Genet. Dev. (2002) vol 12 (1), 22-29).

One of the tumor promoting functions of TGF-beta is that it contributes to invasion and metastasis by inducing EMT of cancer cells (Heldin CH, Vanlandewijck M, Moustakas A., Regulation of EMT by TGF-b in cancer. 2012) vol., 586 (14), 1959-1970).

EMT of these cancer cells is called oncogenic EMT by distinguishing it from non-oncogenic EMT induced by TGF-beta in fibrotic diseases. Recent in vivo studies have shown that inhibitors of TGF-beta or TGF-beta receptors decrease the metastatic and invasive properties of various cancer models, and this reduction in metastasis and invasive potential is likely to inhibit EMT activation (Yang et al., 2003). Inhibition of breast cancer metastasis by a novel inhibitor (Fang Y, Chen Y, Yu L, Zheng C, Qi Y, Li Z, Yang Z, Zhang Y, Shi T, of TGF-b receptor I. (2013) J. Natl. Cancer Inst., vol. 105 (1), 47-58).

TGF-beta binds to the type I and II receptors present in the cell membrane and induces type I serine / threonine kinase activity to transmit signals into cells (Shi Y, Massague J., Mechani, TGF-beta signaling from cell membrane to the nucleus (2003) Cell vol. 113 (6) 685-700).

The physiological responses of cells through TGF-beta signaling are mainly driven by the expression of genes at the transcription level, and the role of transcriptional regulatory proteins called Smad as signaling mediators involved in this process is important.

Smad proteins include Smad2 and Smad3, which function through binding to activated TGF-beta receptors, and are sometimes referred to as R-Smad. Smad4 carries the function of transporting these proteins into the nucleus through complexing with activated R-Smad and is called common Smad (Shi Y, Massague J., Mechani, TGF-beta signaling from cell membrane to the nucleus. 2003) Cell vol. 113 (6) 685-700).

Inhibitory Smad, Smad7, is induced by TGF-beta and binds to type I receptor and inhibits its function, forming a negative feedback loop of TGF-beta signaling.

Smad3, which is one of R-Smad, is known to play an important role in the total activity of TGF-beta in cancer cells (Vincent T, Neve EP, Johnson JR, Kukalev A, Rojo F, Albanell J, Pietras K, Virtanen I, Philipson L, Leopold PL, Crystal RG, Herreros AG, Moustakase, Pettersson RF, Fuxe J., A SNAIL1 and Smad3 / 4 transcriptional repressor complex promotes TGF-beta mediated epithelial-mesenchymal transition Biol., 11 (8), 943-950).

In particular, Smad3 plays an important regulatory role in the expression and function of transcriptional regulatory proteins such as Snail, Slug, and ZEB1, which induce the expression of mesenchymal marker genes as key regulators of TGF-beta induced EMT processes. The domain of the Smad3 protein can be largely divided into three domains: mad homology (MH) 1, MH2 and Linker domains. The MH1 domain plays a role in DNA binding and translocation into the nucleus, and the MH2 domain plays an important role in the induction of transcriptional activity of the target gene regulatory region and its interaction with other transcriptional activator proteins (co-activators).

The Linker domain is involved in the regulation of Smad3 function through cross-talk with other signaling pathways and through phosphorylation of Threonine 179, Serine 203, Serine 208 and Serine 213 residues present in these small linker domains (Matsuzaki K. et al. , Smad phosphoisoform signaling specificity: the right place at the right time (2011) Carcinogenesis vol. 32 (11), 1578-1588). Although phosphorylation of these residues has been reported to be important for TGF-beta-induced EMT and cancer cell migration and invasion in cancer cells, the role of phosphorylation of each amino acid residue has not been reported.

Kaempferol (3,5,7-trihydroxy-2- (4-hydroxyphenyl) -4H-1-benzopyran-4-one) was found in edible plants such as tea, broccoli, cabbage, kale, beans and tomato (AE), a review of the dietary flavonoids, kaempferol on human health and cancer chemoprevention. (2013) Food Chem. vol., 138 (4), 2099-2107).

[Prior Patent Literature]

Korean Patent Application No. 10-2006-0021797

The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a novel anticancer drug candidate substance.

Another object of the present invention is to provide an antitumor candidate that inhibits the migration of TGF-beta-induced EMT and cancer cells.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for anticancer chemotherapy comprising a compound represented by the following general formula (1) as an active ingredient.

[Chemical Formula 1]

In one embodiment of the present invention, the cancer is preferably lung cancer, but is not limited thereto.

The present invention also provides a composition for inhibiting epithelial-mesenchymal transition (EMT) induced by TGF-beta containing the compound of the formula (1) as an active ingredient.

[Chemical Formula 1]

The present invention also provides a composition for inhibiting cancer metastasis comprising a compound represented by the following formula (1) as an active ingredient.

[Chemical Formula 1]

The present invention also provides a composition for improving cancer comprising a compound represented by the following formula (1) as an active ingredient.

[Chemical Formula 1]

In addition,

The present invention provides a method for reducing phosphorylation of threonine 179 residue of SMAD (Sma and Mad related proteins) 3 binding domain by TGF-beta1 by treating a cell with a compound of Formula 1 below.

[Chemical Formula 1]

In one embodiment of the present invention, the cell is preferably a lung cancer cell line, and the compound is more preferably, but not limited to, treating cells in vitro.

The present invention also provides a method for inhibiting TGF-beta induced epithelial-mesenchymal transition (EMT) in a cell by treating a cell with a compound of the following formula (1).

[Chemical Formula 1]

The present invention also provides a method for inhibiting TGF-beta-induced MMP (matrix metalloproteinase) -2 expression in a cell of a compound of the following formula (I).

[Chemical Formula 1]

The cancer diseases which can be prevented or treated by the composition of the present invention are preferably lung cancer, but it is preferable that the cancer is lung cancer, but it is not limited to lung cancer, colon cancer, cervical cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, Endometrial carcinoma, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, endometrial cancer, endometrial cancer, endometrial cancer, endometrial cancer, endometrial cancer, endometrial cancer, endometrial cancer, endometrial cancer, Renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, renal pelvic carcinoma, Spinal cord tumor, brainstem glioma, pituitary adenoma, and the like.

The composition of the present invention may be formulated into various forms such as powders, granules, tablets, capsules, oral formulations such as suspensions, emulsions, syrups and aerosols, injections of sterilized injection solutions, and the like, And can be administered via various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Examples of suitable carriers, excipients or diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The composition of the present invention may further comprise a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, an antiseptic, and the like.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, Mix and formulate. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the oral liquid preparation include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included .

Examples of preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. 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, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the compound of the present invention may vary depending on the age, sex, and body weight of the patient, and is generally 0.01 to 100 mg, preferably 0.1 to 10 mg per kg of body weight, 3 times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

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

In the present invention, "administration" means providing a predetermined substance to a patient in any suitable manner, and the administration route of the compound of the present invention may be administered orally or parenterally . In addition, the composition of the present invention can be administered using any device capable of delivering an effective ingredient to a target cell.

The term "subject" in the present invention means an animal including, but not limited to, a human, a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a rat, Mammal in one embodiment, and human in another embodiment.

The term "therapeutically effective amount " used in connection with the active ingredient in the present invention means the amount of benperin, its pharmaceutically acceptable salt or derivative thereof, which is effective in treating or preventing a subject disease.

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

Examples of anticancer agents that can be included in the pharmaceutical composition of the present invention include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphate, The compounds of the present invention include cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin cisplatin and carboplatin; Antitumor antibiotics (anti-cancer antibiotics)

but are not limited to, dactinomycin: actinomycin D, doxorubicin: adriamycin, daunorubicin, idarubicin, mitoxantrone, plicamycin, mitomycin, C Bleomycin; And plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, and iridotecan, Iridotecan, and the like, but are not limited thereto.

Hereinafter, the present invention will be described.

When the TGF-beta was treated with human lung cancer cell line A549 for 36 hours, the epithelial cell morphology changed into spindle-shaped cells compared to the untreated condition. These changes were observed when Kaempferol (25 mM) (FIG. 2A).

The expression of E-cadherin and the expression of N-cadherin and smooth muscle alpha actin were significantly reduced by kaempferol in the epithelial to mesenchymal transition (EMT) induced by TGF-beta (FIG. 2B).

The effect of kaempferol on the expression of these EMT markers (E-cadherin, N-cadherin, and Vimentin) was also confirmed by immunofluorescence staining of A549 cells (FIG. 2C).

In cancer cells, EMT is an essential process in the early stages of cancer progression, such as invasion and migration of cancer cells. We investigated whether kaempferol inhibition effect of TGF-beta-induced A549 cancer cells on EMT was similar to that of TGF-beta-induced migration and invasion of cancer cells.

Scratching assay analysis showed that the migration rate of cancer cells increased by TGF-beta was markedly reduced by kaempferol (FIG. 3A).

Trans-well migration assay results also showed the same reduction effect (FIG. 3B).

The effect of kaempferol on the expression of matrix metalloproteinase-2 (MMP-2), which plays an important role in the invasion process of cancer cells, was confirmed by the increase in TGF-beta1-induced MMP-2 expression by kaempferol (FIG. 3C).

The role of Smad3 as a key signaling mediator of EMT induction by TGF-beta1 is well known. Activation of the Smad3 protein requires phosphorylation of C-terminus serine 423/425 residues and translocation into the nucleus through complexation with the Smad4 protein.

However, the phosphorylation of TGF-beta1-induced Smad3 C-terminal residues was not reduced by kaempferol (FIG. 4A) nor did it inhibit migration into the nucleus (FIG. 4B). These results imply that kaempferol inhibits TGF-beta1-induced EMT through other mechanisms without affecting C-terminal phosphorylation and nuclear translocation of Smad3.

There are reports that the binding domain of Smad3 contains important amino acid residues that undergo phosphorylation and that their phosphorylation plays an important role in determining the transcriptional activity of Smad3 protein. The effect of Kaempferol on the phosphorylation of Smad3-linked domains by TGF-beta1 was investigated.

It was confirmed that the phosphorylation of the Thr179 residue of Smad3-linked domain by TGF-beta1 was selectively reduced by Kaempferol treatment (FIGS. 5A and 5B).

We investigated whether phosphorylation of Smad3-linked domain Thr179 is important for induction of EMT by TGF-beta1. Expression of Smad3 T179V mutants in which Smad3 Thr179 residues were substituted with valine in A549 cells decreased expression of PAI-1 and N-cadherin increased by TGF-beta1 (FIG. 5C).

In immunofluorescence staining experiments, expression levels of E-cadherin protein reduced by TGF-beta1 were also restored in A549 cells expressing the Smad3 T179V mutation (FIG. 5D).

A similar Smad3 T179V mutation expression effect was confirmed in a luciferase reporter gene assay assay measuring E-cadherin promoter activity (Figure 5E).

Increased Snail promoter activity by TGF-beta1 was also significantly reduced by kaempferol (FIG. 5F).

These results suggest that the phosphorylation of Smad3-linked domain Thr179 is important for the metastasis activity including EMT induced by TGF-beta1 in lung cancer cells, and kaempferol inhibits the phosphorylation of Thr179, Exhibit this inhibitory activity.

The present invention has been made to confirm the efficacy of kaempferol, which inhibits the metastasis of TGF-beta1, and the phosphorylation of Smad3-linked domain Thr179 as a mechanism of action, in A549, a lung cancer cell line.

Figure 1 shows the chemical structure of Kaempferol,
Figure 2 shows the inhibitory effect of kaempferol on EMT of A549 lung cancer cells induced by TGF-beta1.
FIG. 3 shows the effect of kaempferol on Smad3 C-terminal phosphorylation and migration into nucleus by TGF-beta1,
Figure 4 shows the inhibitory effect of kaempferol on the migration and MMP-2 expression of A549 lung cancer cells induced by TGF-beta1,
Figure 5 shows the significance of Smad3 Thr179 phosphorylation and the inhibitory effect of kaempferol on Smad3 Thr179 phosphorylation in EMT induced by TGF-beta1.

Hereinafter, the present invention will be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example 1: Cell culture

Human lung cancer cell line A549 was obtained from the American Type Culture Collection (Manassas, Va.). The cells were cultured in RPMI 1640 medium supplemented with 2 mM l-glutamine, 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin, and 100 g / ml streptomycin.

Example 2: Immunoblot analysis

Cell contents were resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 5 mM EDTA, 2 mM DTT, 1 mM sodium orthovanadate, glycerophosphate, 1 mM sodium fluoride, 1 mM microcystin, 1 mM phenylmethylsulfonyl fluoride, 5 mg / ml leupeptin, 10 mg / ml aprotinin) at 4 ° C. The cell lysate was purified by centrifugation at 12,000 rpm / min and 4 ° C for 10 minutes. The supernatant was recovered and the total protein concentration was determined using a Bio-Rad protein assay reagent. The same amount of cell protein was heated at 95 ° C for 3 minutes, separated by SDS-PAGE (polyacrylamide gel electrophoresis), and transferred to PVDF (polyvinylidene difluoride) membrane. The membranes were blocked with Tris-buffered saline containing 0.05% (v / v) Tween 20 and 5% (w / v) skim milk for 1 hour and then incubated with appropriate antibodies. Primary antibody conjugation was performed using Supersignal West Pico Chemiluminscent Substrate (Pierce / ThermoScientific, Rockford, Calif.) Using Horseradish peroxidase-attached goat anti-mouse or anti-rabbit immunoglobulin G secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) IL).

Example 3: Indirect immunofluorescence microscopy

Cells were grown on cover slips, rinsed in PBS, fixed with 3.7% formaldehyde in PBS for 10 minutes at 4 ° C and permeabilized with PBS supplemented with 0.5% Triton X-100 at room temperature for 10 minutes. The permeabilized cells were rinsed three times with PBS (PBST) supplemented with 0.5% Tween 20 and blocked with blocking solution (0.1% BSA in PBS, pH 7.4) for 90 minutes. Cells were rinsed 3 times in PBS and over-night incubated at 4 [deg.] C with an anti-Smad3 rabbit polyclonal antibody (1: 200 dilution in PBST; Zymed, Santa Cruz, CA) mixture. After rinsing three times in PBST, the cover slip was incubated at 37 [deg.] C for 1 hour in a 1: 300 diluted Alexa-488-attached anti-rabbit IgG secondary antibody (Molecular Probes) mixture in PBST. Cell nuclei were counter-stained with DAPI (4,6-diamidino-2-phenylindole). The intracellular distribution of the Smad3 protein was analyzed at 488 nm (for Alexa-488) and 340 nm (for DAPI) using confocal fluorescence microscopy (Fluoview, FV-300, Olympus, Melville, NY).

Example 4: Zymography

Kaempferol in cells After 30 min pre-treatment, TGF-b1 is treated for 24 h. Collect cells and medium after 24 hours. Proteins from the cells are used to quantify the proteins in the medium. Make an acryamide gel containing gelatin at a concentration of 20 mg / ml, and add the medium with 2X SDS sample buffer. Separate the proteins present in the medium by SDS PAGE and wash the acrylamide gel twice in a wash buffer (0.5 M Tris / HCl pH 7.5, 2.5% TritonX100) for 30 min. After washing, incubation buffer (0.5 M Tris / HCl pH 7.5, 10 mM CaCl ₂ , 0.15 M NaCl, 0.02 & sodium azide) was reacted at 37 ° C for 18 hours.

After 18 hours, discard the incubation buffer and stain gel for 30 minutes using Commasie blue buffer (0.1% commassie blue, 25% methanol, 0.7% acetic acid). After removal of Commasie blue buffer, the degradation of gelatin by MMP protein was confirmed by using destaining buffer (Methanol: Acetic acid: water = 2: 1: 4).

Example  5: DNA Transfection  And Lucifer Reyes  Essay

Cells to 24-well plates (1 x 10 ⁵ cells / plate), and seeding, the pGL3-E-cadherin or pGL3-luciferase reporter plasmid of Snail 0.5mg T179V with Smad3 expression plasmid and the control plasmid DNA FuGENE 6 reagent on (Roche, Mannheim) according to the manufacturer ' s instructions. All plasmids were cotransfected with Escherichia coli beta-galactosidase (Lac Z) structural gene, 0.2 mg CMV-beta-GAL, a eukaryotic expression vector under transcriptional control of the CMV promoter. Twenty-four hours after the transfection, the cells were either treated or not treated with 5 ng / ml TGF-beta 1 for 24 hours, washed twice with cold PBS and then recovered. Luciferase reporter activity was measured with a luminometer with a dual-Luciferase Reporter Assay System (Promega, Madison, Wis.) And its activity was normalized to the CMV-beta-GAL gene. Transfection experiments were performed in duplicate in separate sets of two independent experiments and the results were averaged.

Example  6: Wounds healing  Essay

Place the cells in a 6-well plate and grow them until the cells are densely populated. When the cells grow densely, use a 200ul tip to scrape the center of the 6well in a straight line. After scraping the cells, wash twice with PBS and add new medium. Then kaempferol was treated 30 minutes before TGF-b1 treatment and cultured for 36 hours. The photographs were taken at 12-hour intervals for confirmation, and finally the cell mobility after 36 hours was confirmed by photographs.

Example  7: Transwell  Move essay

Place the cells in the upper part of the Traswell with 5X10 ⁴ water. The lower part of the transwell is fed with the medium in which the serum exists, and the upper part where the cells are present is cultured in a serum-free medium. Once cells are attached to the transwell, kaempferol is pretreated 30 minutes prior to concentration. After kaempferol treatment 30 minutes, TGF-b1 is treated and cells are cultured for 12 hours. After 12 hours, the cells that have migrated to the lower part of the Trasnwell are stained with methanol for 1 minute. After 1 minute, wash with water for 30 seconds and then hematoxylin for 7 minutes. After hematoxylin staining, wash for 30 seconds and stain Eosin for 1 minute. After Eosin staining, wash for 1 minute and wash immediately for 30 seconds.

After wiping off the water and detached cells, immerse the membrane located under the transwell for a short time in xylene and fix it on the slide glass. The number of transferred cells is observed by a microscope, and the number of transferred cells is measured by taking a photograph of four portions at random.

Claims (16)

delete delete delete delete delete delete delete A method of reducing the phosphorylation of threonine 179 residues of SMAD (Sma and Mad related proteins) 3 binding domain by TGF-beta1 in a corresponding cell in vitro by treating the compound of formula (1)

[Chemical Formula 1] 9. The method according to claim 8, wherein the cell is a lung cancer cell line. delete delete delete delete delete delete delete

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