KR20180036458A - Method for screening antitumor agent using galangin - Google Patents

Abstract

One aspect of the present invention relates to a method for screening an anticancer agent, comprising: a step of contacting a cell comprising an Smad3 protein or a fragment thereof, or a nucleotide sequence encoding the same with galangin and a test substance, respectively; a step of measuring whether the Smad3 protein or fragment thereof is phosphorylated in a sample containing the cell; a step of comparing a degree of phosphorylation measured in a sample containing the galangin-contacted cells and a sample containing cells in contact with the test substance; and a step of selecting a test substance having a lower degree of phosphorylation as measured relative to the galangin-contacted cells. In addition, the present invention relates to a kit for screening anticancer substances, comprising: a cell comprising the Smad3 protein or fragment thereof, or the nucleotide sequence encoding the same; and a reagent for detecting the phosphorylation of the Smad3 protein or fragment thereof in a sample containing said cell.

Description

[0001] METHOD FOR SCREENING ANTITUMOR AGENT USING GALANGIN [0002]

And a method for screening anticancer substances using gallanazine, which is known to have an anticancer effect.

Identification of key factors involved in the functional transformation of TGF-β as a cancer-inducing factor in cancer suppressor and identification of its mechanism of action are recognized as important in the development of therapeutic agents for cancer development and metastasis. According to recent reports, the linker of Smad3 protein is a target of various phosphorylated proteins in the cell, suggesting that the phosphorylation may be important in controlling the transcription activity of Smad3 protein. Recently, the present inventors have observed that TGF-β-induced apoptosis and cell growth inhibition signal transduction systems are further activated when phosphorylation of Smad3 protein is inhibited in the linker of TGF-β signal transduction pathway , Thereby promoting cancer growth and cell death, and inhibiting the formation of cancer stem cells.

Accordingly, the present inventors searched for a natural material for controlling or inhibiting phosphorylation at the linker of Smad3 protein, and confirmed antitumor effect. Recently, it has been reported that kaempferol, which is a natural substance of flavonoids, regulates phosphorylation at the junction of Smad3 protein and has cancer-preventing effect and cancer-metastasis-inhibiting effect. In order to develop a method for screening substances having anticancer effect using galantine as a control group, it was confirmed whether or not gallantrine, which is structurally very similar to camphorol, has an anticancer effect.

In one aspect, there is provided a method for screening a cell, comprising contacting a cell comprising a Smad3 protein or fragment thereof, or a nucleotide sequence encoding the same, Measuring whether the Smad3 protein or fragment thereof is phosphorylated in the sample containing the cell; Comparing the degree of phosphorylation measured in a sample containing cells with the galactan-contacted cells and a sample containing cells in contact with the test substance; And a step of selecting a test substance having a lower degree of phosphorylation as measured relative to the scavenging activity.

Another aspect is a cell comprising a Smad3 protein or fragment thereof, or a nucleotide sequence encoding it; And a reagent for detecting phosphorylation of a Smad3 protein or fragment thereof in a sample containing the cell.

In one aspect, there is provided a method for identifying a cell, comprising contacting the cell comprising the Smad3 protein or a fragment thereof, or a nucleotide sequence encoding the same, Measuring whether the Smad3 protein or fragment thereof is phosphorylated in the sample containing the cell; Comparing the degree of phosphorylation measured in a sample containing cells with the galactan-contacted cells and a sample containing cells in contact with the test substance; And a step of selecting a test substance having a lower degree of phosphorylation as measured relative to the scavenging activity.

The Smad3 refers to a protein functioning in a cell. In the present specification, the 'protein' is used interchangeably with 'polypeptide'. The 'amino acid sequence' of the protein is also used interchangeably with the 'peptide sequence'. The 'nucleotide sequence encoding' the Smad3 protein means that the codon sequence encoding the amino acid sequence of the Smad3 protein is included, and one or more codons encoding one amino acid residue may be present, And may include all the numbers in the case of the codon listing.

The term " contacting " means that a specific substance binds to the cell membrane or cell wall or is injected into the cell. The contacting step may include, for example, adding to the medium containing the cell, May be injected directly into the containing tissue, or, if applied to an individual, be injected into the blood to reach the desired cells.

The 'step of measuring the phosphorylation status' may be performed by using an antibody specific to phosphorylated specific amino acid residues, measuring the degree of radiation using a radiation-labeled phosphate group, or using an LC-MS / MS sequencing method. For example, when an LC-MS / MS sequence is used, the degree of phosphorylation or the phosphorylated amino acid residue can be analyzed by measuring the mass change of a specific amino acid residue and the mass of the phosphorylated group while sequencing the peptide sequence. Also, in the case of using a radiation-labeled phosphoric acid group, a chemiluminescent enzyme, or a fluorescent substance, the degree of phosphorylation of the Smad3 protein can be measured by measuring the exposure dose, the amount of emitted light, or the amount of fluorescence.

The step of selecting a test substance having a lower degree of 'measured phosphorylation degree' means that the degree of phosphorylation of Smad3 protein, particularly the degree of phosphorylation of Smad3 protein, is inhibited as shown in the present specification, and at the same time, And when cancer is treated, the tumor cell toxicity, tumor cell death or cancer cell metastasis inhibition phenomenon appears together with the degree of inhibition of the phosphorylation of Smad3 protein. Therefore, when the test substance is contacted with cells, In particular, when the degree of phosphorylation of the Smad3 protein is decreased, the test substance may be expected to have tumor cytotoxicity, tumor cell death or cancer cell metastasis inhibitory effect such as gallantin. Therefore, the test substance judged to have a lower degree of phosphorylation measured according to the above method can be selected as an anticancer substance. The term " phosphorylation " means that a phosphate group is bonded to a functional group of a specific amino acid residue, or the functional group is substituted with a phosphate group, and can be produced by an intracellular enzyme such as kinase. The amino acid residue capable of phosphorylation may be, for example, threonine or serine. The term 'inhibition' means having a lower level than the standard level and can mean 'inhibition', 'blocking' or 'downward regulation'.

In the 'Smad3 protein or a fragment thereof, or a cell comprising the nucleotide sequence encoding the Smad3 protein or fragment thereof', the 'nucleotide' may be an endogenous nucleic acid material inherently present in the cell, and may be an extrinsic exogenous nucleic acid material.

In one embodiment, the Smad3 protein used in the screening method of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 1. Accordingly, in this case, the 'Smad3 protein or fragment thereof, or a cell comprising the nucleotide sequence encoding the same' may refer to a cell comprising a nucleotide sequence of SEQ ID NO: 2 or a fragment thereof as an intracellular nucleic acid material. In this case, the phosphorylation may occur at any one or more selected from the group consisting of amino acids 179, 208, 213, 423 and 425 in the amino acid sequence of SEQ ID NO: 1. Referring to FIG. 2C, it can be seen that phosphorylation is inhibited by amino acid residues 179, 208, 213 and 423/425 of the polypeptide sequence of the Smad3 protein by galactan having an anticancer effect. Therefore, it is possible to screen for a substance having anticancer effect by judging whether phosphorylation is inhibited to the amino acid residue. In addition, since phosphorylation is most strongly inhibited in the phosphorylation inhibition effect of the Smad3 protein of gallantin is the 179th threonine amino acid residue in the amino acid sequence of SEQ ID NO: 1, the degree of inhibition of phosphorylation of the amino acid residue 179 It may be more advantageous to screen an experimental material having an anticancer effect above or below the threshold.

 In one embodiment, in the method of screening for an anticancer substance, the phosphorylation may be performed at an amino acid residue of an amino acid sequence corresponding to a linker of the Smad3 protein. In the amino acid sequence of SEQ ID NO: 1, the amino acid residues 179, 208 and 213 correspond to the linker of the Smad3 protein. Galanzin inhibits the phosphorylation of the connecting amino acid residues of the Smad3 protein, The test substance has an anticancer effect when the specific test substance reduces the degree of phosphorylation of at least one of amino acid residues 179, 208 and 213 in the amino acid sequence of SEQ ID NO: 1, It can be judged that it is caused by a mechanism similar to that of Gin.

In one embodiment, the Smad3 used in the screening method of the present invention may be derived from a human. Smad3 proteins may have the same function in a variety of genetically distinct individuals, but may have different nucleotide or peptide sequences. Thus, since the polypeptide sequence may be different depending on the individual, Smad3 of such an individual may have a different sequence from the polypeptide sequence of SEQ ID NO: 1. Since the nucleotide sequence encoding the Smad3 protein may contain a homologous gene, the nucleotide sequence encoding the Smad3 protein may be different from that of the Smad3 derived from the genetically different individual.

In one embodiment, in the method of screening of the present invention, the cell may be a tumor cell. Wherein said tumor cell is selected from the group consisting of lung cancer, liver cancer, esophageal cancer, stomach cancer, colon cancer, small bowel cancer, pancreatic cancer, oral cancer, brain tumor, thyroid cancer, pituitary cancer, kidney cancer, cervical cancer, sarcoma, prostate cancer, Lymphoma, skin cancer, psoriasis, and fibroadenoma. The present inventors have found that when the phosphorylation is inhibited in at least one amino acid residue selected from the group consisting of amino acid residues 179, 208 and 213 of the amino acid sequence of the Smad3 protein in prostatic cancer and pancreatic cancer cells, And increased tumor cell apoptosis or tumor cell growth inhibition. Thus, at least in prostate cancer and pancreatic cancer cells, gallantine inhibits the phosphorylation of the binding site of the Smad3 protein to the amino acid sequence and induces the inhibition of the phosphorylation, thereby having antitumor or cancer metastasis inhibitory effect. Also, if the substance inhibits phosphorylation at one or more amino acid residues selected from the group consisting of amino acid residues 179, 208 and 213 of the amino acid sequence of the Smad3 protein-binding site of the tumor, the substance has an antitumor effect on a specific tumor The present invention also relates to a method of treating cancer in a patient suffering from cancer such as lung cancer, liver cancer, esophageal cancer, stomach cancer, colon cancer, small bowel cancer, pancreatic cancer, oral cancer, brain tumor, thyroid cancer, parathyroid cancer, Cancer, lymphoma, skin cancer, psoriasis, and fibroadenoma.

In one embodiment, in the method of screening of the present invention, the step of contacting the galangan and the test substance, respectively, may further comprise the step of adding TGF- ?. When galantamine acts together with TGF-ss, the rate of decrease in phosphorylation level is increased and the degree of tumor cytotoxicity, inhibition of tumor cell growth and inhibition of cancer cell metastasis is increased, so that the degree of inhibition of the detected phosphorylation is large Or when the detection signal is weak, the step of comparing the degree of phosphorylation measured by its synergy in the case of TGF-β treatment together can be made more advantageous. In addition, since it is possible to judge whether or not the anticancer effect has an interaction with TGF-beta, it can be expected that the candidate test substance has a mechanism similar to that of gallantin, which is advantageous for screening anticancer substances.

In one embodiment, the step of screening the test substance in the screening method of the present invention further includes a step of measuring the degree of phosphorylation measured in a sample containing cells in contact with the test substance in comparison with the sample containing the cells in contact with the galangan If the test substance is low, the test substance may be selected as an anticancer substance. It has been observed that when guanine is contacted with tumor cells, it inhibits the phosphorylation of Smad3 protein in tumor cells, thereby inhibiting tumor cell growth, inhibiting tumor cell death and inhibiting cancer cell metastasis. Therefore, when the phosphorylation of the Smad3 protein (particularly the connecting site) of the candidate test substance is more strongly inhibited than that of gallanthin, it can be selected as an anticancer substance because it is judged to have an anticancer effect comparable to or similar to gallanthin.

In another embodiment, the step of screening the test substance in the screening method of the present invention comprises contacting the test substance with TGF-? Relative to a sample containing cells in contact with the galangan and TGF-? If the degree of phosphorylation measured in the sample is lower, the test substance may be selected as an anticancer substance. When galantamine acts in combination with TGF-β, the rate of decrease in phosphorylation level is increased, and tumor cytotoxicity, inhibition of tumor cell growth and inhibition of cancer cell metastasis are increased, so that the degree of inhibition of phosphorylation detected is not significantly different, In the case of TGF-β treatment, the phosphorylation of Smad3 protein (especially the junction) of the candidate test substance is more strongly inhibited than that of gallantin by comparing the degree of phosphorylation measured by its synergy, It can be selected as an anticancer substance because it is judged to have an anticancer effect superior to that of similar or similar antigens.

In one embodiment, in the method of screening of the present invention, said anti-cancer effect may comprise inhibiting cancer cell growth. Referring to FIG. 6, the expression of c-myc, a protein involved in cancer cell growth, was found to be dose-dependent and significantly decreased in tumor cells contacted with galantin. Thus, in one embodiment, the screening method can be selected as an anticancer substance by comparing the degree of reduction of c-myc expression using the candidate candidate test substance as a control group. Therefore, when the candidate test substance significantly reduced c-myc or contacted with tumor cells at the same concentration compared to the decrease amount of c-myc expression of gallantin, , The candidate test substance can be selected as an anticancer substance because it is judged that the growth of tumor cells can be more effectively inhibited with respect to the tumor cells than at least with respect to the galangan.

Another aspect is a cell comprising a Smad3 protein or fragment thereof, or a nucleotide sequence encoding it; And a reagent for detecting phosphorylation of a Smad3 protein or fragment thereof in a sample containing the cell.

The kit for screening an anticancer substance may be provided in the form of a kit including a packaging unit having one or more reaction reagents. The kit may also contain one or more of the following items: a cell containing a buffer, instructions for use, a Smad3 protein or fragment thereof, or a nucleotide sequence encoding it, an appropriate concentration of galangin, or a Smad3 protein or fragment thereof Reagents for detecting phosphorylation. The kit may comprise containers of reagents mixed in suitable proportions to carry out the methods described herein. The reaction reagent vessels preferably contain a unit quantity of reaction reagent so as to omit the measuring step when performing the method. In one embodiment, the reagent for detecting phosphorylation may comprise phosphoric acid comprising phosphorus, which is a radioactive isotope, when measuring the radiation in the method of use of the kit, and measuring the amount of luminescence The kit may further include a chemiluminescent enzyme, or the kits may further include a fluorescent substance when measuring the amount of fluorescent light. In another embodiment, in the kit for screening anticancer agents, the reagent is selected from the group consisting of amino acids 179, 208, 213, 423 and 425 of the amino acid sequence of SEQ ID NO: 1 when the kit uses, for example, the Western blotting method And an antibody specific to any one or more selected from the group consisting of: In addition, when the Western blotting method comprises measuring radiation, the reagent comprises a phosphorylated secondary antibody comprising the phosphorylation isotope (phosphorylation of 179, 208, < RTI ID = 0.0 > 213, 423, and 425 amino acids of the amino acid sequence of SEQ ID NO:

In one aspect, there is provided a method for identifying a cell, comprising contacting the cell comprising the Smad3 protein or a fragment thereof, or a nucleotide sequence encoding the same, Measuring whether the Smad3 protein or fragment thereof is phosphorylated in the sample containing the cell; Comparing the degree of phosphorylation measured in a sample containing cells with the galactan-contacted cells and a sample containing cells in contact with the test substance; And selecting a test substance having a lower degree of phosphorylation as measured relative to the scavenging ability can be used to rapidly screen an anticancer substance having anticancer effect similar or superior to that of gallan.

A cell comprising a Smad3 protein or fragment thereof, or a nucleotide sequence encoding it, in another aspect; And an anti-cancer substance screening kit containing a reagent for detecting phosphorylation of the Smad3 protein or a fragment thereof in a sample containing the cell can be conveniently used to screen for an anticancer substance having anticancer effect similar or superior to that of gallan.

Figure 1 shows the chemical formulas of camphorol and gallanzin.
FIG. 2A shows the phosphorylation change of the Smad3 protein induced by camphorol in the prostate cancer cell line to a specific amino acid residue, and FIG. 2B shows the change in the specific amino acid residue of the Smad3 protein induced by the galantin in the prostate cancer cell line FIG. 2C shows the result of Western blotting for confirming the inhibitory effect of phosphorylation of camptol and cleavage on specific amino acid residues in pancreatic cancer cell line, and FIG. 2D is a diagram showing the phosphorylation site of Smad3 protein.
FIG. 3 shows a comparison of inhibition of cell growth according to the concentration of camphorol and galantin in prostate cancer cell lines.
FIG. 4 shows a comparison of inhibition of cell growth according to the log concentration of camphorol and gallan in the pancreatic cancer cell line.
FIG. 5 shows the interaction of TGF-β and galantin in prostate cancer and pancreatic cancer cell line in terms of cell viability.
FIG. 6 shows Western blotting results for confirming the interaction effect of TGF-β on galantin and galantin in the prostate cancer cell line as the level of protein expression of apoptotic markers.
FIG. 7 shows FACS results for confirming the interaction effect of TGF-beta on galantin and galantin in prostate cancer cell lines.
FIG. 8 is a graphical representation of FACS results for confirming the role of TGF-beta on galantin and galantin in prostate cancer cell lines.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Gallantine Smad3  Confirmation of phosphorylation inhibition effect on protein phosphorylation site (Western blotting)

Western blotting was performed to confirm the phosphorylation inhibitory effect on the phosphorylation site of Smad3 protein-linked phosphorylation site of gallantin in prostate cancer and pancreatic cancer cell line at the protein expression level.

Specifically, the human prostate cancer cell line PC3M was treated with galantine at 0, 10, 25, and 50 μM, followed by incubation for 30 minutes, and 50 μM of camphor (KF) and galantine (Gal) were added to human pancreatic cancer cell line SNU2491 After incubation, the cells were incubated for 30 minutes. Then, 5 ng / ml of TGF-β1 was added to each cell culture and incubated for 1 hour. The cell lines were washed with PBS (phosphate-buffered saline) and lysed in a solution (20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X- Cocktail) was added to each of the above cell lines. Then, the cell lysate was heated with addition of SDS sample buffer, and subjected to SDS-PAGE electrophoresis to separate the intracellular proteins by size. The protein was then transferred from the SDS-PAGE gel to the PVDF blotting membrane using transfer buffer and voltage. 3% BSA (bovine serum albumin) solution was added to the PVDF blotting membrane, followed by incubation for one hour to block. The primary antibody specific for the amino acid sequence of each phosphorylated Smad3 protein was added to the 3% BSA solution Lt; RTI ID = 0.0 > 4 C < / RTI > for 12 hours. The blocked PVDF blotting was washed three times with TBST (tri-buffered saline-tween20) buffer, immersed in 3% BSA supplemented with secondary antibody, incubated for 1 hour at room temperature, and washed three times with TBST buffer. Then, chemiluminescence by the peroxidase of the secondary antibody was induced, and the amount of protein expression in the cell was qualitatively analyzed by a chemiluminescence imaging device.

As a result, as shown in Figs. 2A and 2B, both camphor and gallan were found to affect the degree of phosphorylation of the Smad3 protein to a specific amino acid residue, and in particular, specific amino acid residues in which the degree of phosphorylation was significantly decreased were Smad3 It was confirmed that the amino acid sequence was the amino acid sequence 179 threonine amino acid residue.

In addition, as shown in FIG. 2C, the degree of phosphorylation of each amino acid residue is also greater in the case of treatment with gallanazine than in camphorol. In particular, the amino acid sequence corresponding to the linkage portion of Smad3 protein has amino acid residue 179 Of the phosphorylation of the cells.

On the other hand, the degree of phosphorylation in TGF-β1 treatment seemed to be rather increased, but phosphorylation decreased rapidly when treated with galantamine, and in the case of treatment with tumor cells by combination of galantamine and TGF- Suggesting that it may be advantageous to treat TGF-β1 with either galantine or a candidate agent to observe Smad3 phosphorylation inhibition, since the differences are in sharp contrast.

Example  2: Gallantine  Tumor cell cytotoxicity and TGF -β MTT  analysis)

In this example, MTT assay was performed to determine the cytotoxicity and the effect of TGF-β on the tumor cells of gallantin in prostate cancer and pancreatic cancer cell line.

Specifically, by a dehydrogenase action, the yellow water-soluble substrate MTT tetrazolium is replaced with a purple water-insoluble MTT formazan (3- (4,5-dimethylthiazol-2-yl) Tetrazolium bromide) by measuring the ability of mitochondria to reduce the cell viability. 96 Plate wells were inoculated with 5 x 10 ³ of each of the prostate cancer and pancreatic cancer cell lines, and the camelol and galantin were added to the cell culture at concentrations of 0 to 100 μM, respectively. In the experimental group of TGF-β1, TGF-β1 5 ng / ml was further added and incubated for 48 hours. A 1/10 MTT solution was then added to the culture and incubated for 4 hours. The medium was carefully removed from the culture, and 200 μl of DMSO was added to each well. After incubation for 30 minutes, the absorbance was measured using a 540 nm wavelength in a microplate detector.

As a result, as shown in Fig. 3, cell viability was lower in both prostatic cancer cells and pancreatic cancer cell lines when galantin was treated than camphorol. As shown in FIG. 4, when the concentration of compound of camphor and gallan is logarithmically expressed, the degree of cell survival is significantly different. Therefore, a remarkable effect on the growth inhibitory ability of tumor cells, particularly prostate cancer and pancreatic cancer cells As shown in FIG.

In addition, as shown in FIG. 5, the inhibitory effect of galantamine on proliferation of prostate cancer and pancreatic cancer cell line was further increased when TGF-β was added together. Therefore, it can be shown that gallantin has an inhibitory effect on tumor cell growth in tumor cells, particularly, prostate cancer and pancreatic cancer cells, and TGF-β has a synergistic effect on such effects.

This means that gallantine is excellent as a control in screening for anticancer substances, and it can be compared with the results of galantin to determine whether candidate anticancer substances have cell growth inhibitory effect and interaction with TGF-beta1. It is also suggested that the treatment with TGF-β1 may increase the difference in the effect, which may be advantageous for screening candidates having antitumor effect.

Example  3: Gallantine  Confirmation of cell killing effect

In this example, the effect of gallantine on tumor cell death was examined in a prostate cancer cell line.

(One) Western Blotting

To investigate the cytotoxic effect of gallantin on prostate cancer cell line by analyzing the protein expression level of apoptotic factor.

Specifically, the human prostate cancer cell line PC3M was treated with 0, 50, and 100 μM of galantamine, respectively, followed by incubation for 30 minutes. Then, 5 ng / ml of TGF-β1 was added to each cell culture and incubated for 48 hours. Then, the cells were treated as in Example 1, and the primary antibody specific to each cell death marker was added to the 3% BSA solution, followed by incubation at 4 ° C for 12 hours. The blocked PVDF blotting was washed three times with TBST (tri-buffered saline-tween20) buffer, immersed in 3% BSA supplemented with secondary antibody, incubated for 1 hour at room temperature, and washed three times with TBST buffer. Then, chemiluminescence by the peroxidase of the secondary antibody was induced, and the amount of protein expression in the cell was qualitatively analyzed by a chemiluminescence imaging device.

As a result, as shown in FIG. 6, it was found that the amount of zalli-PARP, which is an apoptosis marker, was increased, and the degree was further increased when TGF-β was added. In addition, the amount of truncated-caspase3 was increased, and at the same time Smad3 phosphorylation (especially the 179th threonine) was also decreased, which is further increased by the addition of TGF-β. Thus, when gallanzin has a tumor cell killing effect in prostate cancer and Smad3 phosphorylation is inhibited, tumor cell killing effect is significant, and TGF-beta is involved in it. Furthermore, even though phosphorylation is likely to increase again in the TGF-beta1 treatment, the phosphorylation is reduced and the increase / decrease in the amount of the phosphorylation is increased in the treatment of gallantin. Therefore, And it is possible to more clearly judge whether or not TGF-beta has an anti-cancer effect by treating TGF-beta together.

(2) FACS  analysis

To investigate the effect of Galantin on tumor cell death in prostate cancer cell line using flow cytometry.

Specifically, the human prostate cancer cell line was contacted with 50 μM of galantamine alone or with TGF-β1, followed by incubation for 48 hours. When apoptosis occurs, the phosphatidylserine inside the cell membrane is exposed, and the principle of binding of Annexin V protein and cell apoptosis proceeds. When the permeability of the cell membrane increases when necrosis occurs, PI penetrates into the cell and stains the nucleus The cell death was confirmed by the Annexin V / PI staining method using the principle. The number of stained cells was measured with a flow cytometer to compare the degree of cell death in each group.

As a result, as shown in Fig. 7, it can be shown that gallantin is able to induce apoptosis, and the degree of TGF-beta treatment is further increased. Therefore, it is possible to more clearly determine whether or not TGF-beta has an anti-cancer effect by treating TGF-beta together.

In addition, as shown in FIG. 8, the effect of galantin and TGF-beta is further increased in the late apoptotic cell death, leading to apoptosis from the initial apoptosis, and ultimately, the combination of galantin, Indicating that there is a significant effect on cell death.

The results of the above examples show that galantzine is very similar in structure to camphoryl but more effectively inhibits the phosphorylation of the Smad3 protein at its linkage and is superior to camphorol in inhibiting growth of cancer cells and has an anticancer effect superior to camphorol . Therefore, it can be said that it can be a good control for screening anticancer substances.

In addition, it was confirmed that galinzin significantly inhibited phosphorylation at the junction of Smad3 protein in the cell death or growth inhibition signal transduction in combination with TGF- Therefore, it is possible to screen whether the test substance has an excellent anticancer effect and cancer cell metastasis inhibitory effect by using the galantinase as a control, and to test whether the phosphorylation at the junction of the Smad3 protein is inhibited by using galantine as a control, In the case of TGF-β1, the selected experimental material would have an effect similar to that of Gallan in Smad3. In addition, in the case of evaluating the inhibition of phosphorylation in tumor cells, the expression of tumor cell death factor, or the inhibition of tumor cell growth in a combination with TGF-β1, galantin can be advantageously used as a control for screening antitumor factors will be.

<110> Advanced Institutes of Convergence Technology <120> METHOD FOR SCREENING ANTITUMOR AGENT USING GALANGIN <130> PN115515 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 425 <212> PRT <213> Homo sapiens <400> 1 Met Ser Ser Ile Leu Pro Phe Thr Pro Pro Ile Val Lys Arg Leu Leu   1 5 10 15 Gly Trp Lys Lys Gly Glu Gln Asn Gly Gln Glu Glu Lys Trp Cys Glu              20 25 30 Lys Ala Val Lys Ser Leu Val Lys Lys Leu Lys Lys Thr Gly Gln Leu          35 40 45 Asp Glu Leu Glu Lys Ala Ile Thr Thr Gln Asn Val Asn Thr Lys Cys      50 55 60 Ile Thr Ile Pro Arg Ser Leu Asp Gly Arg Leu Gln Val Ser His Arg  65 70 75 80 Lys Gly Leu Pro His Val Ile Tyr Cys Arg Leu Trp Arg Trp Pro Asp                  85 90 95 Leu His Ser His His Glu Leu Arg Ala Met Glu Leu Cys Glu Phe Ala             100 105 110 Phe Asn Met Lys Lys Asp Glu Val Cys Val Asn Pro Tyr His Tyr Gln         115 120 125 Arg Val Glu Thr Pro Val Leu Pro Pro Val Leu Val Pro Arg His Thr     130 135 140 Glu Ile Pro Ala Glu Phe Pro Pro Leu Asp Asp Tyr Ser His Ser Ile 145 150 155 160 Pro Glu Asn Thr Asn Phe Pro Ala Gly Ile Glu Pro Gln Ser Asn Ile                 165 170 175 Pro Glu Thr Pro Pro Pro Gly Tyr Leu Ser Glu Asp Gly Glu Thr Ser             180 185 190 Asp His Gln Met Asn His Ser Met Asp Ala Gly Ser Pro Asn Leu Ser         195 200 205 Pro Asn Pro Met Ser Pro Ala His Asn Asn Leu Asp Leu Gln Pro Val     210 215 220 Thr Tyr Cys Glu Pro Ala Phe Trp Cys Ser Ile Ser Tyr Tyr Glu Leu 225 230 235 240 Asn Gln Arg Val Gly Glu Thr Phe His Ala Ser Gln Pro Ser Met Thr                 245 250 255 Val Asp Gly Phe Thr Asp Pro Ser Asn Ser Glu Arg Phe Cys Leu Gly             260 265 270 Leu Leu Ser Asn Val Asn Arg Asn Ala Ala Val Glu Leu Thr Arg Arg         275 280 285 His Ile Gly Arg Gly Val Arg Leu Tyr Tyr Ile Gly Gly Glu Val Phe     290 295 300 Ala Glu Cys Leu Ser Asp Ser Ala Ile Phe Val Gln Ser Pro Asn Cys 305 310 315 320 Asn Gln Arg Tyr Gly Trp His Pro Ala Thr Val Cys Lys Ile Pro Pro                 325 330 335 Gly Cys Asn Leu Lys Ile Phe Asn Asn Gln Glu Phe Ala Ala Leu Leu             340 345 350 Ala Gln Ser Val Asn Gln Gly Phe Glu Ala Val Tyr Gln Leu Thr Arg         355 360 365 Met Cys Thr Ile Arg Met Ser Phe Val Lys Gly Trp Gly Ala Glu Tyr     370 375 380 Arg Arg Gln Thr Val Thr Ser Thr Pro Cys Trp Ile Glu Leu His Leu 385 390 395 400 Asn Gly Pro Leu Gln Trp Leu Asp Lys Val Leu Thr Gln Met Gly Ser                 405 410 415 Pro Ser Ile Arg Cys Ser Ser Val Ser             420 425 <210> 2 <211> 1278 <212> DNA <213> Homo sapiens <400> 2 atgtcgtcca tcctgccttt cactcccccg atcgtgaagc gcctgctggg ctggaagaag 60 ggcgagcaga acgggcagga ggagaaatgg tgcgagaagg cggtcaagag cctggtcaag 120 aaactcaaga agacggggca gctggacgag ctggagaagg ccatcaccac gcagaacgtc 180 aacaccaagt gcatcaccat ccccaggtcc ctggatggcc ggttgcaggt gtcccatcgg 240 aaggggctcc ctcatgtcat ctactgccgc ctgtggcgat ggccagacct gcacagccac 300 cacgagctac gggccatgga gctgtgtgag ttcgccttca atatgaagaa ggacgaggtc 360 tgcgtgaatc cctaccacta ccagagagta gagacaccag ttctacctcc tgtgttggtg 420 ccacgccaca cagagatccc ggccgagttc cccccactgg acgactacag ccattccatc 480 cccgaaaaca ctaacttccc cgcaggcatc gagccccaga gcaatattcc agagacccca 540 ccccctggct acctgagtga agatggagaa accagtgacc accagatgaa ccacagcatg 600 gacgcaggtt ctccaaacct atccccgaat ccgatgtccc cagcacataa taacttggac 660 ctgcagccag ttacctactg cgagccggcc ttctggtgct ccatctccta ctacgagctg 720 aaccagcgcg tcggggagac attccacgcc tcgcagccat ccatgactgt ggatggcttc 780 accgacccct ccaattcgga gcgcttctgc ctagggctgc tctccaatgt caacaggaat 840 gcagcagtgg agctgacacg gagacacatc ggaagaggcg tgcggctcta ctacatcgga 900 ggggaggtct tcgcagagtg cctcagtgac agcgctattt ttgtccagtc tcccaactgt 960 aaccagcgct atggctggca cccggccacc gtctgcaaga tcccaccagg atgcaacctg 1020 aagatcttca acaaccagga gttcgctgcc ctcctggccc agtcggtcaa ccagggcttt 1080 gaggctgtct accagttgac ccgaatgtgc accatccgca tgagcttcgt caaaggctgg 1140 ggagcggagt acaggagaca gactgtgacc agtaccccct gctggattga gctgcacctg 1200 aatgggcctt tgcagtggct tgacaaggtc ctcacccaga tgggctcccc aagcatccgc 1260 tgttccagtg tgtcttag 1278

Claims (15)

Contacting the cell comprising the Smad3 protein or fragment thereof, or the nucleotide sequence encoding it, with the galantine and the test substance, respectively;
Measuring whether the Smad3 protein or fragment thereof is phosphorylated in the sample containing the cell;
Comparing the degree of phosphorylation measured in a sample containing cells with the galactan-contacted cells and a sample containing cells in contact with the test substance; And
Selecting a test substance having a lower degree of phosphorylation as measured relative to the scavenging activity. The method of claim 1, wherein the Smad3 protein is a polypeptide having the amino acid sequence of SEQ ID NO: 1. The method according to claim 1, wherein the phosphorylation occurs at any one or more selected from the group consisting of amino acids 179, 208, 213, 423, and 425 in the amino acid sequence of SEQ ID NO: The method according to claim 1, wherein the phosphorylation occurs at an amino acid residue of an amino acid sequence corresponding to a linker of the Smad3 protein. The method according to claim 4, wherein the amino acid residue is at least one amino acid residue selected from the group consisting of amino acids 179, 208 and 213 in the amino acid sequence of SEQ ID NO: 1. 5. The method of claim 4, wherein the amino acid residue is amino acid residue 179 in the amino acid sequence of SEQ ID NO: 1. The method according to claim 1, wherein the Smad3 is derived from a human. The method according to claim 1, wherein the cell is a tumor cell. The method of claim 1, wherein contacting the galangan and the test substance, respectively, further comprises adding TGF-ss. The method according to claim 1, wherein the step of selecting the test substance comprises the step of: when the degree of phosphorylation measured in the sample containing cells contacting the test substance is lower than that of the sample containing the cells in contact with the galangan, &Lt; / RTI &gt; [Claim 9] The method according to claim 9, wherein the step of selecting the test substance comprises measuring the degree of phosphorylation measured in a sample containing cells in contact with the test substance and TGF- [beta] relative to a sample containing cells in contact with the galangan and TGF- And if lower, the test substance is selected as an anti-cancer substance. 11. The method of claim 10 and 11, wherein the anti-cancer effect comprises inhibiting tumor cell growth. [Claim 12] The method according to claim 12, wherein the inhibition of tumor cell growth is judged by comparing the degree of decrease in expression of c-myc. A cell comprising a Smad3 protein or fragment thereof, or a nucleotide sequence encoding the same; And
And a reagent for detecting the phosphorylation of the Smad3 protein or fragment thereof in a sample containing the cell. The reagent according to claim 14, wherein the reagent is selected from the group consisting of an anticancer substance screening comprising one or more antibodies specific to any one or more selected from the group consisting of amino acids 179, 208, 213, 423 and 425 of the amino acid sequence of SEQ ID NO: For the kit.

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