KR20180036461A - Composition comprising galangin for inhibition of cancer metastasis and for treating cancer - Google Patents

Abstract

The present invention relates to a composition for treating cancer, containing a substance which inhibits phosphorylation of a Smad3 protein as an active ingredient. The present invention further relates to a method for treating cancer, comprising a step of administering, to an individual, a composition for treating cancer containing the substance which inhibits phosphorylation of the Smad3 protein as the active ingredient.

Description

Technical Field [0001] The present invention relates to a composition for inhibiting cancer metastasis and cancer,

A composition comprising gallanazine that inhibits phosphorylation at the junction of the Smad3 protein, and a method of inhibiting cancer metastasis and cancer using the composition.

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. Therefore, it was investigated whether gallanzine, which is structurally very similar to camphorol, could function as an anticancer substance, and developed a composition for treating cancer.

One aspect of the present invention relates to a composition for treating cancer comprising a substance that inhibits phosphorylation of Smad3 protein as an active ingredient.

Another aspect relates to a method of treating cancer, comprising administering to a subject a composition for treating cancer comprising as an active ingredient a substance that inhibits phosphorylation of the Smad3 protein.

One aspect of the present invention provides a composition for treating cancer comprising as an active ingredient a substance that inhibits phosphorylation of Smad3 protein.

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'.

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 composition may comprise the above materials and their pharmaceutically acceptable salts. There is provided a use of a composition comprising a pharmaceutically acceptable salt. The term " pharmaceutically acceptable salt " refers to a concentration that is relatively non-toxic to a patient and has a beneficial effect that is harmless, such that the side effects caused by the salt are any organic or inorganic species of the compound of formula S that does not impair the beneficial efficacy of the compound of formula Means an addition salt. Examples of the inorganic acid include hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid and phosphoric acid. Examples of the organic acid include citric acid, acetic acid, lactic acid, maleic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, tartaric acid, succinic acid, malonic acid, succinic acid, malonic acid, glutamic acid, aspartic acid, oxalic acid, P-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid. These salts also include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, the acid addition salt may be selected from the group consisting of acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camylate, citrate, eddylate, Hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, malate, glucoside, gluconate, gluconate, glucuronate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / Hydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydroxyacetate, Lactate, stearate, succinate, tartrate, tosylate, trifluoroacetate Diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, and the like. Lt; / RTI > hydrochloride or trifluoroacetate.

In one embodiment, the Smad3 protein in the cancer treatment composition of the present invention may be a polypeptide having the amino acid sequence of SEQ ID NO: 1. Thus, 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: Referring to FIG. 2C, it can be seen that phosphorylation is inhibited by amino acids residues 179, 208, 213 and 423/425 of the polypeptide sequence of the Smad3 protein by galantamine.

In one embodiment, the phosphorylation in the composition for treating cancer of the present invention may be that occurring at the amino acid residue of the amino acid sequence corresponding to the 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 Smad3 protein, As a result, a substance that inhibits the phosphorylation of the Smad3 protein has a strong anticancer effect such as gallantin. In particular, when the phosphorylation of 179th threonine is inhibited in the amino acid sequence of SEQ ID NO: 1, as in the case of treatment with galantzin, inhibition of tumor cell growth, tumor cell apoptosis, or inhibition of cancer metastasis appears most strongly, The substance inhibiting the phosphorylation of the 179 threonine in the amino acid sequence of SEQ ID NO: 1 has an anticancer substance.

In one embodiment, the Smad3 in the composition for treating cancer 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.

In one embodiment, the cell in the composition for treating cancer of the present invention may be a tumor cell.

The present invention also relates to a method for the treatment of cancer such as lung cancer, liver cancer, esophageal cancer, stomach cancer, colon cancer, small bowel cancer, pancreatic cancer, oral cancer, bone cancer, brain tumor, thyroid cancer, papillary cancer, kidney cancer, cervical cancer, , Multiple myeloma, myelodysplastic syndrome, or hematologic cancers such as glioblastoma, Hodgkin's lymphoma or lymphoma such as non-Hodgkin's lymphoma, Behcet's disease, skin cancer, psoriasis and fibrosarcoma. The present inventors have confirmed 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, cancer metastasis is suppressed Tumor cell death or tumor cell growth inhibition was increased. Therefore, at least the inhibition of the phosphorylation of the binding site of the Smad3 protein in the prostate cancer and pancreatic cancer cells to the above amino acid sequence, and the galangin inducing the phosphorylation inhibition have antitumor or cancer metastasis inhibitory effects on prostate cancer and pancreatic cancer cells . 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, the substance in the cancer treating composition of the present invention may be gallantine.

According to the results of the present inventors' studies, gallanz inhibited the phosphorylation of the Smad3 protein and thus produced a strong anticancer effect. In particular, in the amino acid sequence of SEQ ID NO: 1 corresponding to the linkage of Smad3 protein, amino acid residues 179, 208 and 213 Inhibited the phosphorylation of 179 threonine most strongly and inhibited tumor cell toxicity, inhibited tumor cell growth and inhibited cancer cell metastasis. Therefore, a composition comprising galantine as an active ingredient can be used as a composition for inhibiting cancer metastasis and cancer.

In one embodiment, the anticancer effect in the composition for treating cancer of the present invention may include inhibiting cancer cell growth. 6, the expression of c-myc, a protein involved in cancer cell growth, was found to be dose-dependent and significantly decreased in the tumor cells contacted with galantamine. Thus, the cancer treatment effect of the composition may be caused by inhibiting tumor cell growth, and the inhibition of tumor cell growth may be due to a decrease in c-myc expression.

In one embodiment, in the composition for treating cancer of the present invention, the composition may further comprise TGF-ss.

According to the results of the present inventors' studies, when galantzin is combined with TGF-β, the cytotoxicity, inhibition of tumor cell growth, and inhibition of cancer cell metastasis are increased, so that phosphorylation at the junction of Smad3 protein, such as galantin or gallan, When the inhibiting substance is treated in combination with TGF-β, synergy of the complex can provide a stronger antitumor effect.

Another aspect provides a method for treating cancer, comprising administering to a subject a composition for treating cancer comprising as an active ingredient a substance that inhibits phosphorylation of a Smad3 protein.

Such administration can be carried out in various formulations for oral administration or for parenteral administration such as intravenous, intraperitoneal, subcutaneous, subcutaneous, epithelial or muscular administration. In the case of formulation, the fillers, extenders, binders, wetting agents, Release agents, surfactants, and the like.

Solid formulations for oral administration may include tablets, patients, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. As the liquid preparation for oral administration, suspensions, solutions, emulsions or syrups may be used. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, .

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, 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. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

In one embodiment, in the method of treating cancer, the subject may be a mammal. The mammal may be a human, and the effective dose of the compound of the present invention on the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity.

There is provided a composition for treating cancer comprising a substance inhibiting phosphorylation of Smad3 protein according to a certain aspect as an active ingredient and administering the composition to a subject, And can cure cancer of an individual.

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 2C shows the result of Western blotting to confirm the inhibitory effect on the phosphorylation of camptol and cleavage specific amino acid residues in the pancreatic cancer cell line and FIG. 2D shows 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. Human pancreatic cancer cell line SNU2491 was treated with 50 μM each of galantamine and 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- It can be understood that the Smad3 phosphorylation inhibitor may be advantageous in terms of the effect when the composition is used as a composition for treating cancer by a combination of TGF-β1, and that Gallanthin has a beneficial effect as the Smad3 phosphorylation inhibitor.

Example 2: Tumor cell cytotoxicity of gallantin and confirmation of effect of TGF-beta (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 suggests that galantine has an excellent effect as a composition for treating cancer in at least prostate cancer and pancreatic cancer, and can be more advantageous in the cancer treatment effect when it is treated with a combination of TGF-β.

Example 3: Confirmation of cell death effect of galantine

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

(1) 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. Moreover, even though phosphorylation is likely to increase again in the TGF-beta1 treatment, the phosphorylation is reduced and the increase / decrease in the degree of increase and decrease is increased at the time of treatment with gallan, so that a substance inhibiting phosphorylation of Smad3 protein can be used as a composition for treating cancer. Galangin can be used as a substance that inhibits phosphorylation of a protein, and it can be a composition having an excellent cancer treatment effect, especially when it is used in combination with TGF-beta.

(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 seen that galantamine, which is a substance inhibiting the phosphorylation of Smad3 protein, can induce apoptosis, and the degree of this increase is further increased when TGF-beta is treated. Therefore, it is possible to more clearly determine whether or not TGF-beta has an anti-cancer effect by treating TGF-beta together.

As shown in Fig. 8, the effect of the galangan and TGF-beta, which are substances that inhibit the phosphorylation of Smad3 protein, is further increased in the late apoptosis, leading to cell death from early apoptosis, And TGF-b have a significant effect on tumor cell death.

As a result of the above example, it is found that the substance that inhibits the phosphorylation of Smad3 protein has at least cancer treatment effect in prostate cancer and pancreatic cancer, and TGF-beta has a synergistic effect on the effect. In addition, galactan, which is a substance that inhibits phosphorylation of Smad3 protein, is structurally very similar to camphoryl, but inhibits the phosphorylation of Smad3 protein more effectively, has excellent ability to inhibit cancer cell growth more than camphorol, It can be seen that Jean is excellent.

In addition, since it seems that inhibition of phosphorylation of Smad3 protein and inhibition of phosphorylation of Smad3 protein junction, particularly 179 threonine residue, play an important role in inhibiting tumor cell apoptosis or tumor cell growth, the 179th amino acid sequence of Smad3 protein A substance which inhibits phosphorylation of threonine residues will have a remarkable effect as a composition for treating cancer as compared with other Smad3 phosphorylation inhibitors.

<110> Advanced Institutes of Convergence Technology <120> COMPOSITION COMPRISING GALANGIN FOR INHIBITION OF CANCER          METASTASIS AND FOR TREATING CANCER <130> PN115801 <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)

A composition for treating cancer, comprising a substance that inhibits phosphorylation of Smad3 protein as an active ingredient. The composition according to claim 1, wherein the Smad3 protein has the amino acid sequence of SEQ ID NO: 1. The composition for treating cancer 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: 1. The composition for treating cancer according to claim 1, wherein the phosphorylation occurs at an amino acid residue of an amino acid sequence corresponding to a linker of Smad3 protein. [Claim 4] The composition according to claim 4, wherein the amino acid residue is an 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 composition according to claim 4, wherein the amino acid residue is 179 threonine in the amino acid sequence of SEQ ID NO: 1. The composition according to claim 1, wherein the Smad3 is derived from a human. The composition according to claim 1, wherein the cell is a tumor cell. The composition according to claim 8, wherein the tumor is prostate cancer or pancreatic cancer. The composition according to claim 1, wherein the composition inhibits the growth of tumor cells. [Claim 11] The composition according to claim 10, wherein the tumor cell growth inhibition is by a decrease in expression of c-myc. The composition according to claim 1, wherein the substance is gallantin. 13. The composition for treating cancer according to claim 12, wherein the composition further comprises TGF-beta. A method of treating cancer, comprising administering to the subject a composition according to claim 1. 15. The method of claim 14, wherein the subject is a mammal.

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